Receptors for fibroblast growth factors

ABSTRACT

A fibroblast growth factor (FGF) receptor including a basic fibroblast growth factor receptor has been purified. Various forms have bee identified including soluble forms lacking any transmembrane segment. DNA sequences encoding full-length fibroblast growth factor receptors and polypeptides comprising a portion of an FGF-R ligand-binding domain have been isolated and sequenced. These DNAs include DNAs encoding for a basic FGF-R and a human FGF-R and are operably linked to control sequences and expressed in a culture of a compatible host transformed, transfected or electroporated by a cloning vehicle containing the DNA sequence. The invention also comprises antibodies to the receptor, methods of synthesizing the growth factor receptor proteins, methods for providing analogs of the fibroblast growth factor receptors. Methods for evaluating compositions which promoter or inhibit fibroblastic growth factors and compositions which are agonistic or antagonistic to fibroblast growth factor receptors are also provided. Diagnostic and therapeutic uses are described.

This application is a Division of application Ser. No. 07/834,311 filedFeb. 13, 1992, the U.S. National Phase of PCT/US90/03830, filed Jul. 61990, which is a continuation-in-part application of commonly assignedpatent application U.S.S.N. 07/377,003 filed on July 6, 1989, nowabandoned which is hereby incorporated herein by reference.

This invention was made in part with government support under grantcontract No. HL-07192 and under grants RO1 HL-32898 and P01 HL-43821-01,all awarded by the National Institutes of Health. The government mayhave certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to receptors for growth factors,specifically to the fibroblast growth factor receptor (FGF-R). Moreparticularly, it provides various purified fibroblast growth factorreceptor proteins, nucleic acids encoding the receptor proteins, methodsfor the production of purified FGF-R proteins, proteins made by thesemethods, antibodies against these proteins, and diagnostic andtherapeutic uses of these various reagents.

BACKGROUND OF THE INVENTION

Polypeptide growth factors are mitogens that act on cells byspecifically binding to receptors situated at the plasma membrane. Thesereceptors usually have three major identifiable regions. The first is anextracellular region which contains the domain that binds thepolypeptide growth factor (i.e. the ligand-binding domain). The secondregion is a transmembrane region and the third is an intracellularregion. Many of these receptors contain a tyrosine kinase domain in theintracellular region.

The fibroblast growth factor receptor (FGF-R) proteins bind to a familyof related growth factor ligands, the fibroblast growth factor (FGF)family. This family of growth factors are characterized by amino acidsequence homology, heparin-binding avidity, the ability to promoteangiogenesis and mitogenic activity toward cells of epithelial,mesenchymal and neural origin.

The FGF family includes the following seven known FGFs:

(1, 2) acidic FGF (aFGF) and basic FGF (bFGF) (D. Gospodarowicz et al.,Mol. Cell. Endocrinol., 46:107 (1986);

(3) the int-2 gene product (R. Moore et al., EMBO. J., 5:919 (1986);

(4) the hst gene product or Kaposi's sarcoma FGF (K. J. Anderson et al.Nature, 332:360 (1988); M. Taira et al., Proc. Natl. Acad. Sci. USA,84:2980 (1987));

(5) FGF-5 (X. Zhan et al., Mol. Cell. Biol., 8:3487 (1988)); and

(6) keratinocyte growth factor (J. S. Rubin et al., Proc. Natl. Acad.Sci. USA, 86:802 (1989)).

(7) FGF-6 (I. Marics, et al., Oncogene 3:335 (1989)).

The actions of acidic and basic FGF are mediated through binding to highaffinity cell surface receptors of approximately 145 and 125 kDa (G.Neufeld and D. Gospodarowicz, J. Biol. Chem., 261:5631 (1986)).

The reference of Imamura et al., “Purification of Basic FGF Receptorsfrom Rat Brain,” Biochem. Biophys. Res. Communications, 155:583 (Sep.15, 1988) discloses the purification of nanogram amounts of a basic FGFreceptor (bFGF-R) from rat brain.

While genes encoding a number of growth factor receptors have beenmolecularly cloned (e.g., mouse PDGF receptor, Yarden et al., Nature,323:226 (1986), no clone has previously been identified as encoding afibroblast growth factor receptor (FGF-R). Using antiphosphotyrosineantibodies to screen λgt11 cDNA expression libraries, a 2.5 kilobasecDNA encoding a novel tyrosine kinase gene, designated bek (bacteriallyexpressed kinase), was isolated from a mouse liver cDNA library. (S.Kornbluth et al., “Novel Tyrosine Kinase Identified by PhosphotyrosineAntibody Screening of cDNA Libraries”, Mol. Cell. Biol. No. 8, 541(1988)). The bek sequence did not contain a transmembrane region andtherefore could not be identified as a growth factor receptor. Anotherprotein tyrosine kinase gene designated flg (fms-like-gene) was isolatedfrom a human endothelial cell cDNA library by hybridization underrelaxed stringency with a v-fms oncogene probe. (M. Ruta et al., “ANovel Protein Tyrosine Kinase Gene Whose Expression is Modulated DuringEndothelial Cell Differentiation”, Oncogene, 3:9 (1988)). Those authorscould not identify a transmembrane region in their isolated sequence andtherefore hypothesized that flg encodes a cytoplasmic tyrosine kinase.

The purified and cloned chicken bFGF and human bFGF receptors of thisinvention have amino acid sequence similarity with the bek and flgclones in the regions which have been isolated. However, both the bekand flg sequences reported were incomplete and there was no recognitionof their function as FGF binding receptors. Moreover, the prior reportsfailed to recognize many of the structural and functional featuresdescribed in the present invention.

Members of the FGF family appear to have roles in tissue development,tissue repair, maintenance of neurons and in the pathogenesis ofdisease. Aberrant expression of FGF may cause cell transformation by anautocrine mechanism. Moreover, FGFs may enhance tumor growth andinvasiveness by stimulating blood vessel growth in the tumor or byinducing production of proteins such as plasminogen activator. However,identification of the components involved and understanding of themechanisms and interactions involved remain woefully incomplete.

Purified FGF receptors and fragments, and isolated DNA sequencesencoding defined FGF receptors and defined fragments (e.g., theligand-binding domain) will greatly accelerate the understanding offibroblast growth factor functions. Antibodies against specific anddefined regions of the FGF receptor also become available. Thesereagents will find both diagnostic and therapeutic uses in theaforementioned processes. The present invention fulfills these and otherneeds.

SUMMARY OF THE INVENTION

The present invention provides purified fibroblast growth factorreceptor (FGF-R) proteins, nucleic acids encoding FGF-R proteins,methods for the production of purified FGF-R proteins, purified proteinsmade by these methods, antibodies against these proteins and fragments,and diagnostic and therapeutic uses of these reagents. Notably, thepresent invention provides soluble and secreted forms of the receptorsexhibiting an unusual receptor structure.

The present invention provides a method for modifying in vivo afibroblast growth factor receptor modulated activity comprisingadministering to a patient an amount of a fibroblast growth factorreceptor blocking agent effective to inhibit fibroblast growth factorbinding to said fibroblast growth factor receptor. Typically, the agentwill be a fragment of a human fibroblast growth factor receptor, e.g., afragment produced in a cell transformed with a nucleic acid containingat least about 15 bases of a sequence selected from the group consistingof:

a) a DNA sequence in FIGS. 3 or 4;

b) a sequence encoding a polypeptide of FIGS. 3, 4 or 7; and

c) a sequence substantially homologous to a sequence of FIGS. 3 or 4.

The fragment will often be a fibroblast growth factor receptorextracellular domain without a tyrosine kinase region.

Alternatively, a method is provided for inhibiting binding between afibroblast growth factor and a fibroblast growth factor receptor in asolution. This method will contain a step of combining an FGF-R peptide,e.g., a peptide homologous in sequence to a sequence described in FIGS.3, 4 or 7 to a solution or medium containing fibroblast growth factorand fibroblast growth factor receptor, usually native fibroblast growthfactor receptor. Such methods will be useful in vitro, after employinglabeled FGF-R peptide in assay procedures.

Compositions containing a soluble FGF-R polypeptide having between aboutfive and two hundred contiguous amino acids from a human FGF-Rextracellular domain are described. In one embodiment, the polypeptidecontains at least about 80 amino acids from residues 1 to 287 of a humanfibroblast growth factor receptor of FIG. 7 or an IgII or IgIII domain,or both. In alternative embodiments, the IgII domain will have about 7contiguous amino acids from residues 85 to 141 of a human sequence ofFIG. 7 or may contain a carboxy-terminal sequence substantiallyhomologous to the 79 amino acid sequence from residues 222 to 300 of asoluble human protein of FIG. 7. Particularly preferred polypeptidesconsist essentially of the h4 or h5 sequences (FIG. 7).

A further aspect of the invention is a fibroblast growth factor receptorcomposition containing a substantially pure polypeptide of less thanabout 85 KDa comprising a fibroblast growth factor-binding domain. Thepolypeptide may be soluble or may specifically possess a signal segment,an IgI segment, an acidic segment, an IgII segment, an IgIII segment, anIgIIIT segment, or a transmembrane segment. Preferred embodiments willbe homologous to a sequence described in FIGS. 3, 4 or 7 or will includeat least about 30 amino acids of each of both IgII and IgIII domains.The polypeptide can be one polypeptide chain in a multi-chain complex ofproteins. A chicken fibroblast growth factor receptor is one preferredembodiment.

The present invention embraces isolated nucleic acids encoding humanfibroblast growth factor receptor proteins which substantially lack anintracellular domain. Such a nucleic acid will usually exhibit asequence homologous to an IgII domain described in FIG. 7, or mayinclude a substantially full length IgII domain. The nucleic acid willusually also have a signal segment, an IgI segment, an acidic segment,an IgIII segment, an IgIIIT segment, a transmembrane segment, or atyrosine kinase segment, and will preferably correspond to a sequencedescribed in FIGS. 3, 4 or 9. A particularly preferred embodiment is anucleic acid encoding a receptor native to a human. The nucleic acidsmay be operably linked to a transcription promoter sequence and mayfurther be incorporated into expression vectors suitable for productionof recombinant FGF-R peptide.

Also included are isolated nucleic acids encoding a soluble humanfibroblast growth factor receptor, preferably one homologous to h4 orh5. Protein products made by expressing such an isolated nucleic acidare provided.

A method is provided for making these proteins of newly recognizedutility, e.g., fibroblast growth factor receptor activity, said methodcomprising expressing an isolated nucleic acid. Products produced bythis method are now also available.

Additional methods are provided for making fibroblast growth factorreceptor peptides by transforming a cell with a nucleic acid of at leastabout 21 bases of a sequence selected from the group consisting of:

a) a DNA sequence in FIGS. 3, 4 or 9;

b) a sequence encoding a polypeptide of FIGS. 3, 4 or 7; and

c) a sequence substantially homologous to a sequence of FIGS. 3, 4 or 9.

Other methods for producing an antibody against a fibroblast growthfactor receptor fragment are described, including a step of producing anantibody against a polypeptide epitope homologous to a sequence of atleast six contiguous amino acids described in FIGS. 3, 4 or 7. Theepitopes of most interest will be those from a signal segment, an IgIsegment, an acidic segment, an IgII segment, an IgIII segment, or anIgIIIT segment.

As a diagnostic use, these reagents provide a method for measuring afibroblast growth factor or a fibroblast growth factor receptor in atarget sample, said method comprising the steps of:

combining said target sample with a fibroblast growth factor receptorsegment; and

determining the extent of binding between said segment and said sample.

This invention also provides a transformed cell capable of expressing apolypeptide homologous to at least a portion of a human fibroblastgrowth factor receptor. A preferred embodiment is where the cellexpresses a polypeptide homologous to substantially the entire membranebound or soluble form of a human fibroblast growth factor receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 compares the binding of various derivatives of FGF to FGF-R. FIG.1(A) is a graph showing the percent binding inhibition of ¹²⁵I-labeledbFGF. FIG. 1(B) is an autoradiograph of bFGF cross-linked Swiss 3T3cells subjected to gel electrophoresis.

FIG. 2(A) is an autoradiograph of cross-linked chicken membranefractions and WGA eluates subjected to gel electrophoresis. FIG. 2(B) isa silver stained gel showing pure FGF receptor resulting from anaffinity purification performed on the WGA-Sepharose 4B column chickenembryo eluate shown in FIG. 2(A).

FIGS. 3A, B, C and D shows the nucleotide and amino acid sequence of achicken bFGF receptor.

FIGS. 4A, B, C, D, and E show the nucleotide and amino acid sequence ofa human FGF receptor.

FIG. 5(A) represents an autoradiograph of a northern blot of chicken RNAprobed with a full length cDNA chicken bFGF receptor under highstringency conditions. FIG. 5(B) represents an autoradiograph of aprimer extension of chicken mRNA subjected to electrophoresis on anacrylamide sequencing gel.

FIG. 6 is a schematic of a chicken bFGF receptor indicating the (solidblock) acidic domain; (cross-hatched block) transmembrane region;(flecked block) tyrosine kinase domain; (S), position of the SH cysteineresidues (in contrast to the S designation of Table I); (W), position oftryptophan residue with respect to the first cysteine residue in theIg-like domain.

FIGS. 7A and B provides an amino acid sequence comparison of variousdifferent FGF receptor forms. The amino acid sequences of 4 humanreceptor forms are shown in comparison to a chicken FGF receptorsequence. Sequences which differ from the chicken FGF receptor sequenceare outlined in open boxes. Transmembrane sequences are underlined.These DNA sequences are in GenBank/EMBL data bases under the followingaccession numbers: h2 is M34185, h3 is M34186, h4 is M34187, and h5 isM34188.

FIG. 8 provides a schematic representation of various different FGFreceptors. The following structural features are identified: hydrophobicputative signal sequence (solid boxes), the highly acidic region (openboxes), transmembrane domain striped boxes), kinase 1 and kinase 2domains (stippled boxes), and the divergent region of h4/h5 (zigzagline). Asterisks indicate the position at which h2 and h4 contain thesequence ArgMet, the chicken receptor contains a single Asn residue, andh3 and h5 contain no corresponding residues. Triangles indicate theposition at which h3 contains a Glu residue and all other receptor formscontain a Lys residue. The numbers at the top of the figure indicate thedegrees of amino acid identities between similar domains of the h2 humanreceptor and the chicken receptor.

FIG. 9 presents a comparison of various human FGF receptor genomicsequences with deduced amino acid sequences of FGF receptor cDNA clones.The sequence of a human genomic fragment obtained by PCR is shown incomparison to human and chicken cDNA sequences. A 1 kb intron separatesgenomic sequences encoding the Ig-like (Ig) domain and the highly acidicregion. Dashed lines represent continuous sequence with no gaps. Thededuced amino acid sequence shown for the chicken FGF receptor beginswith the initiator methionine residue (1) and ends with the acidicregion (EDDDDEDD; amino acids 125-132 in c1 FGF-R). The amino acidsequence shown for the human h2 FGF receptor begins with the initiatormethionine residue (1) and ends with the acidic region (EDDDDDDD; aminoacids 37-44 in h2).

FIG. 10 shows crosslinking of acidic or basic FGF to receptors in cellstransfected with FGF receptor cDNAs. L6 cells (5×10⁵) transfected withthe cFGFR/pSV7d expression construct (lanes 1, 2, 7, and 8), theh2FGFR/pSV7d expression construct (lanes 3, 4, 9, and 10), or withvectors alone (lanes 5, 6, 11, and 12) were incubated with 0.1 pmoles of¹²⁵1-aFGF (lanes 1-6) or ¹²⁵I-bFGF (lanes, 7-12) in the presence orabsence of a 200-fold excess of unlabeled aFGF (lanes 2, 4, and 6) orbFGF (lanes 8, 10, and 12). Binding was performed for 30 minutes at 37°C. Cells were then washed twice with ice cold DME H21 containing 20 mMHEPES pH 7.4, 0.2% gelatin, and twice with ice cold PBS. Disuccinimidylsuberate (DSS) was added to a final concentration of 0.15 mM andcrosslinking was allowed to proceed for 15 minutes at 4° C. Samples wereresuspended in sample buffer then subjected to SDS PAGE followed byautoradiography.

FIG. 11 illustrates acidic and basic FGF induction of a ⁴⁵Ca⁺⁺ effluxfrom Xenopus oocytes injected with RNA encoding a chicken FGF receptoror the h2 human FGF receptor. The graphs show ⁴⁵Ca⁺⁺ efflux from oocytesinjected with chicken FGF receptor RNA (A and C, open squares), human h2RNA (B and D, open squares), human h3 RNA (B and D, solid triangles) orwater (A-D, solid squares). Injected oocytes were incubated with ⁴⁵CaCl₂for 3 hours at 19° C. and then washed extensively. Groups of 5 oocyteswere placed in individual wells of a 24 well plate and 0.5 ml of mediawas added. At 10 minute intervals, the media was removed for countingand fresh media was added. After 40 minutes, aFGF (panel A and B) orbFGF (panel C and D) were added to a final concentration of 0.5 nM. As apositive control, carbachol was added after 100 minutes. Each data pointrepresents the average of triplicate wells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT OUTLINE I. GeneralDescription

A. FGF-R

1. structural features

a. extracellular domain i. signal sequence ii. Ig domains iii. acidicamino acid region

b. transmembrane segment

c. intracellular domain i. tyrosine kinase ii. insert

2. function

a. bind FGF

b. bind to FGF-R peptide

c. tyrosine kinase activity

B. Physiological Functions

1. cellular

2. tissue differentiation

3. organismal

II. Polypeptides

A. Soluble Forms

B. Truncated Forms

C. Fusion Proteins

D. Genetic Variants (site-directed mutagenesis)

E. Compositions Comprising Proteins

III. Nucleic Acids

A. Isolated Nucleic Acids

B. Recombinant Nucleic Acids

C. Compositions Comprising Nucleic Acids

IV. Methods for Making FGF-R

A. Protein Purification

1. affinity with derivatized FGF

2. various ligands, same receptor

B. Expression of Nucleic Acids

V. Antibodies VI. Methods for Use

A. Diagnostic

B. Therapeutic

I. General Description

A first aspect of the invention provides homogeneous FGF-R peptides.These homogeneous FGF-Rs include a chicken basic fibroblast growthfactor receptor and various human fibroblast growth factor receptors.Homogeneous polypeptides either having FGF-R ligand-binding activity orcomprising a portion of the ligand-binding domain of an FGF-R aredescribed. Notably, the present invention provides homogeneouspolypeptides corresponding to naturally occurring FGF-binding proteinshaving unexpected structural features. One class provides solubleproteins lacking a transmembrane segment, another class providesproteins possessing both a transmembrane segment and a tyrosine kinasedomain. Both of these classes have an unexpected extracellular domainstructure shorter than the corresponding chicken FGF-R. Experimentaldata indicating that a single receptor binds various FGF types is alsodescribed.

A second aspect of the invention provides isolated DNA sequences. Thesesequences encode polypeptides having FGF-R ligand-binding activity,including polypeptides which correspond to naturally occurringfull-length fibroblast growth factor receptors. DNA sequences encoding achicken bFGF-R or encoding various human FGF-Rs (hFGF-R) have beenisolated. Also provided are cloning and expression vehicles containingthe FGF-R encoding sequences. A DNA sequence encoding the full-lengthFGF receptor or an FGF-R polypeptide fragment can be operably linked tocontrol sequences and expressed in a culture of a compatibletransformed, transfected or infected host cells.

Methods of synthesizing growth factor receptor proteins and methods forproviding analogues of the fibroblast growth factor receptors areprovided.

The invention also provides antibodies to defined domains of thereceptor. Still further aspects of the invention include methods forevaluating compositions which are agonistic or antagonistic to ligandand receptor interactions, particularly those which promote or inhibitbinding interactions.

Diagnostic and therapeutic uses for the reagents provided herein arealso described.

A. FGF Receptors

The fibroblast growth factor receptors (FGF-R) are receptors for thefamily of fibroblast growth factors (FGFs), as described above. See alsoP. L. Lee et al., Science 245:57-60, (1989), which is herebyincorporated herein by reference.

The FGF family consists of polypeptide growth factors characterized byamino acid sequence homology, heparin-binding avidity, the ability topromote angiogenesis, and mitogenic activity toward cells of epithelial,mesenchymal, and neural origin. The FGF family includes acidic FGF,basic FGF, the int-2 gene product, the hst gene product (Kaposisarcoma-FGF), FGF-5, the keratinocyte growth factor, and FGF-6. Membersof the FGF family appear to have roles in development, tissue repair,maintenance of neurons, and the pathogenesis of disease. Aberrantexpression of FGFs may cause cell transformation by an autocrinemechanism. Moreover, FGFs may enhance tumor growth and invasiveness bystimulating blood vessel growth into the tumor or by inducing productionof proteases such as plasminogen activator.

The term “ligand” refers to the molecules, usually members of thefibroblast growth factor family, that bind the domains involved in thegrowth factor binding. Also, a ligand is a molecule which serves eitheras the natural ligand to which the receptor binds, or a functionalanalogue which may serve as an agonist or antagonist.

As described herein, a chicken bFGF receptor is characterized by variousidentifiable structural features. The chicken and human FGF-R structuresare generalized to define a structural nomenclature applicable to otherFGF-Rs. General descriptions of protein structure and its relationshipto nucleic acid sequences are discussed in J. D. Watson et al.,Molecular Biology of the Gene, 4th Ed., vols. 1 and 2,Benjamin/Cummings, Menlo Park, Calif., (1987); and B. Alberts et al.,Molecular Biology of the Cell, 2d Ed., Garland, N.Y., (1989), each ofwhich is incorporated herein by reference. Common structural features ofknown FGF-Rs are described, including various naturally occurringsoluble human FGF binding proteins. A human fibroblast growth factorreceptor is a protein either derived from a natural human FGF-R gene, orwhich shares significant structural characteristics peculiar to anaturally occurring human receptor for FGF.

The isolated full-length chicken FGF-R mRNA contains a singlehydrophobic segment similar to a membrane-spanning segment (designatedthe transmembrane segment). The segments of FGF-R amino-proximal to thetransmembrane segment are designated the extracellular domain, while thesegments carboxy-proximal to the transmembrane segment are designatedthe intracellular domain. From the amino-terminus, the extracellulardomain has an NH₂-terminal hydrophobic putative signal sequence, animmunoglobulin-like domain (designated IgI), and acidic segment, asecond immunoglobulin-like domain (designated IgII), and a thirdimmunoglobulin-like domain (designated IgIII). Although variousstructured features may be identified in the external domain of theFGF-R, the most important functional property which defines the domainis the binding to the receptor ligands, e.g., members of the FGF family.As discussed below, this function is correlated with the combinedpresence of IgII and IgIII domains.

The intracellular domain is characterized by the presence of a splittyrosine kinase structural domain and, in the chicken receptor, is about424 residues long. Functionally, this domain is defined by its tyrosinekinase activity, typically modulated by ligand binding to theextracellular domain. A protein substantially lacks an intracellulardomain when it lacks a prototypical intracellular domain, particularlylacking a tyrosine kinase domain.

Besides the chicken receptor, four unique human cDNA clones have beenidentified. These encode previously unknown FGF receptor variants whichcontain only two Ig-like domains. Two of the human clones encodemembrane spanning receptors and two encode putative secreted forms. Boththe forms exhibiting the 3 Ig-like or 2 Ig-like domain structuresmediate biological responsiveness to acidic and basic FGF. Thus, thefirst Ig domain of the 3 Ig domain form may have a function other thanbinding of acidic and basic FGF. The multiple human receptor forms, areidentical in some regions but are highly divergent in other selectedregions of the extracellular domain. Two of the human variant receptors,h4 and h5, are likely to encode a secreted form of the FGF receptor.

A typical FGF-R nucleic acid sequence encodes a transitory NH 2-terminalhydrophobic sequence, which is usually cleaved during the translocationprocess. The classical function of a signal sequence is to direct thenascent polypeptide chain to membrane bound ribosomes, thereby leadingto membrane translocation. However, since the signal sequence istypically removed in the translocation process, the signal sequence isabsent in a mature polypeptide.

The Ig-like domains (Ig domains) are characterized by three mainfeatures: (i) the presence of two characteristic cysteine residues ineach domain; (ii) the presence of a consensus tryptophan residue 11 to12 amino acids on the COOH-terminal side of the first cysteine residuein each Ig-like domain; and (iii) the presence of the consensussequence, DXGXYXC, on the NH₂-terminal side of the second cysteineresidue. The last feature is modified in the cases of the solublereceptor proteins, and substituted with an equivalently sized sequence.

Additional features characteristic of the Ig domains are apparent bothin comparing the domains with one another, and comparing homologousdomains of different receptor molecules. The amino-proximal Ig domainfound in the chicken clone was designated IgI. As the chicken clone hasthree Ig domains, the domains have been numbered from the aminoterminus. As indicated in FIG. 6, the IgI domain includes the 45 aminoacids flanked by a pair of cysteine residues. The chicken IgI domain hasa high homology in sequence with the IgI domain found in the genomicsequence of the human FGF-R. However, the human forms appear to lack adomain corresponding to IgI.

The next Ig domain is designated IgII, and in the chicken receptorincludes 51 amino acids between the two cysteine residues (see FIGS. 3and 6). As described below, this domain, in combination with the IgIIIdomain is involved with ligand binding. The polypeptide sequencehomology of this domain between the chicken and human receptors is quitehigh, as shown by the sequence alignments in FIG. 7. It will be notedthat the human receptors lack an Ig I domain but have IgII and IgIIIdomains. The cysteine residues used to delineate this domain areresidues 176, 89, 87, 89, and 87 on the amino proximal side, and 228,141, 139, 141, and 139 on the carboxy proximal side for the chicken, h2,h3, h4 and h5 receptors, respectively.

The third Ig domain is designated IgIII and in the chicken receptorincludes 63 amino acids between the two cysteine residues. See FIGS. 3and 6. Again, although the human receptors have only two domains, thedomains correspond to IgII and IgIII. In both the chicken and humanforms, the IgIII domain is that closest to the transmembrane segment.The cysteine residues for the chicken, h2, h3, h4 and h5 receptors,respectively, used to delineate this domain are residues 274, 187, 185,187, and 185 on the amino proximal side and residues 339, 252, 250, 253,and 251 on the carboxy proximal side.

The h4 and h5 soluble receptors have a substituted terminal segmentdesignated IgIIIT. This segment is a substituted terminal segmentreplacing part of the membrane bound to IgIII, and is 79 amino acidslong. This sequence corresponds to amino acids 224 and 222 of h4 and h5,respectively, while preserving many of the features found in the IgIIIdomain except of the DSGSYSC. It should be noted, however, the IgIIITsequences are conserved between the soluble forms of the human FGF-R.

Between the first and second immunoglobulin-like domains, the FGFreceptors (shown for the basic FGF-R, but the same FGF-R binds both theacidic and basic FGFS) have a feature not found in other members of theimmunoglobulin superfamily. There is a series of eight consecutiveacidic residues (EDDDDEDD in the case of chicken, and EDDDDDDD in thecase of human) followed by three serine residues and two additionalacidic residues (FIGS. 3 and 7). Although uninterrupted stretches of 7to 35 acidic residues have been described for several intracellularproteins, in particular nuclear proteins, such acidic regions areunusual in the extracellular region of transmembrane receptor proteins.

The 5 receptor species (e.g. the chicken, h2, h3, h4 and h5 forms) alsoexhibit variability at a specific location between the conserved acidicregion and the conserved second Ig-like domain (IgII). The h2 and h4receptor forms contain two amino acids (ArgMet) at positions 59 and 60,while the chicken receptor contains a single amino acid (Asn) at thisposition and the h3 and h5 receptor forms contain no corresponding aminoacids at this position (see asterisks, FIG. 8).

Another unusual feature is the length of the juxtamembrane region, theregion between the membrane spanning segment and the kinase domain. Thisregion is normally conserved among receptor tyrosine kinases. Forexample, the juxtamembrane region is consistently 49 to 51 residues inlength in the receptors for PDGF, CSF-1, epidermal growth factor (EGF),human epidermal growth factor-2 (HER2) and insulin. The FGF receptorswith an intercellular domain have an unusually long juxtamembrane regionof about 87 residues.

The cytoplasmic regions of the amino acid sequences are about 424 and425 residues long, respectively for the chicken and human forms. Thesealso contain a tyrosine kinase sequence (about residues 482 to 759, 395to 672, and 393 to 670, respectively for the chicken, h2, and h3 forms).Overall, the kinase region of the bFGF receptors shares the mostsequence identity (about 51 to 53%) with the PDGF and CSF-1 receptors.The bFGF receptors contain the GXGXXG motif and the conserved lysineresidue (about residue 512) that form part of the adenosine5′-triphosphate (ATP) binding site of tyrosine kinases. The bFGFreceptors also contain the two characteristic tyrosine kinase motifs,HRDLAARNVL and DFGLAR, and a tyrosine (about residues 651, 564 and 562)at the position analogous to the major phosphorylation site ofpp60^(v−src) (about Tyr 416).

The kinase coding sequence of the bFGF receptors, defined by homology toother tyrosine kinases, are split by an insertion of 14 amino acids. Thelength of the insertion in the kinase region is shorter than that foundin the receptors for PDGF and CSF-1 (104 and 70 amino acids,respectively) and is similar to the length of the inserted sequence inthe receptors for insulin and insulin-like growth factor-I.

The FGF-R appears to have three different biological functions. Thefirst is the binding of ligands, usually the FGF proteins or theiranalogues. These ligands or analogues may also serve as either agonistsor antagonists. The ligand binding site is apparently in theextracellular domain. The receptor transduces a signal in response toligand binding, and the result is a ligand modulated activity. As thelikely ligand is a FGF, the signal will ordinarily be FGF-modulated.

A second biological activity relates to the tyrosine kinase enzymaticactivity. This activity is typically activated in response to ligandbinding. However, since the receptors are likely to function in a dimerstate, the intrachain binding interactions may be considered anotherbiological activity which may be mediated by blocking agents. this mayserve as an additional means to modulate FGF-mediation of particularactivities.

B. Physiological Implications

The interactions of FGFs with their receptors cause changes in, onparticular cell types, cell morphology and cell transformation, cellproliferation, cell differentiation, cell senescence, heparinsensitivity, and heparin effects. The in vivo effects of FGF include, inparticular organisms, modulation of various activities, e.g., limbregeneration, lens regeneration, angiogenic effects on both normal andtumor cells, wound healing, adipocyte differentiation, and growth ofvarious neural and myoblast cells. FGFs also exhibit potent angiogenicactivities. It is thought that the angiogenic activity of FGFs is due inlarge part to the chemotactic and mitogenic effects of these factors onendothelial cells. In addition, constitutive expression of FGFs has beenshown to induce cellular transformation in transfected cells, indicatingthat autocrine or paracrine stimulation by FGFs may be involved in tumorformation. These diverse cellular and physiological effects foreshadowthe central importance of these receptor-ligand interactions.

The compositions and cells comprising them can be used for diagnosticpurposes and to study and treat diseases associated with FGF receptors.Cells expressing cloning vehicles containing defined sequences can beused to define specific sites of an FGF receptor necessary for effectinga particular activity. Alternatively, these cells may be useful toassess the ability of a selected receptor to bind different ligands(FGFs and analogues) thereby providing a powerful tool for evaluatingthe potential of drugs for promoting or inhibiting specific FGF-inducedcellular responses.

Cells transfected, injected, infected or electroporated with DNA or MRNAcontaining a full length natural FGF-R sequence will often express thenative or wild type receptor and respond accordingly. Specificconcentrations of a purified receptor or a receptor polypeptide fragmentcan be used to block the binding of the ligand (FGF) to native FGFreceptors. Alternatively, antibodies to the receptor or fragment canhave the same effect.

Homogeneous and defined polypeptides and DNA sequences will find use inraising antibodies. In particular, antibodies against specific regionsof the receptor, e.g., the ligand-binding domain, will find use indiagnostic testing. The reagents FGF-R, FGF-R polypeptides andantibodies to specific regions of the receptor can be used to studyregulation of FGF mediated activities. For example, FGF agonists shouldstimulate blood vessel development, an effect particularly beneficial inwound healing and in the growth of collateral blood vessels in ischemicareas of the heart. FGF antagonists should find use in preventingaberrant angiogenesis as seen in diabetic retinopathy and rheumatoidarthritis or in controlling tumors by blocking proliferation ofvascularization to a tumor.

II. Polypeptides

This invention includes fibroblast growth factor receptor polypeptidesand proteins having FGF-R ligand-binding activity. The receptors of thepresent invention include FGF receptor amino acid sequences such as theamino acid sequences for a chicken bFGF-R and human FGF-R forms as shownin FIGS. 3, 4, and 7. Also included are homologous sequences, allelicvariations, natural mutants, induced mutants, alternatively expressedvariants, and proteins encoded by DNA which hybridize under high or lowstringency conditions, to FGF receptor encoding nucleic acids retrievedfrom naturally occurring material. Closely related FGF-receptorsretrieved by antisera to FGF receptors are also included.

The symbols for the amino acid residues are shown in Table I.

TABLE I Abbreviations for the Amino Acid Residues A, Ala; G, Gly; M,Met; S, Ser; C, Cys; H, His; N, Asn; T, Thr; D, Asp; I, Ile; P, Pro; V,Val; E, Glu; K, Lys; Q, Gln; W, Trp; F, Phe; L, Leu; R, Arg; Y, Tyr; X,any amino acid and Z, termination.

Various new human FGF receptors have been cloned and characterized, asdescribed further below. Of particular note, various shorter forms (h2and h3) and soluble versions (h4 and h5) of FGF receptors have beendiscovered. The soluble proteins (e.g., forms lacking a transmembranesegment) which possess FGF binding capacity indicates that shorter formswill find therapeutic and/or diagnostic uses.

Typically, the fibroblast growth factor receptor peptides of the presentinvention will exhibit at least about 85% homology with thenaturally-occurring receptors in the IgII and IgIII regions, usually atleast about 90% homology, and preferably at least about 95% homology.

In particular, the ligand binding function is localized to theextracellular domain, and the soluble forms retain this particularfunction. Soluble fragments of FGF receptors should be useful insubstituting for or interfering with the functions of the naturallysoluble variants. Alternatively, the soluble forms may interfere withdimerization of FGF receptors, since the receptors may normally be in adimer form. Receptor dimerization may be essential for properphysiological signal transduction.

The human receptors possessing a transmembrane segment are unusual inhaving only the IgII and IgIII of the three Ig domains. The absence ofthe IgI domain indicates that certain functions may be absent in thehuman receptor, or, more likely, that the IgI domain is unnecessary inthe human receptor. Data presented below shows that the IgI domain isnot essential for ligand binding.

As used herein, the terms substantially pure and homogenous describe aprotein which has been separated from components which naturallyaccompany it. Typically, a monomeric protein is substantially pure whenat least about 60 to 75% of a sample exhibits a single polypeptidebackbone. Minor variants or chemical modifications typically share thesame polypeptide sequence. A substantially pure protein will typicallycomprise over about 85 to 90% of a protein sample, more usually willcomprise at least about 95%, and preferably will be over about 99% pure.Normally, purity is measured on a polyacrylamide gel, with homogeneitydetermined by staining. For certain purposes high resolution will beused and HPLC or a similar means for purification utilized. For mostpurposes, a simple chromatography column or polyacrylamide gel will beused to determine purity.

A protein is substantially free of naturally-associated components whenit is separated from the native contaminants which accompany it in itsnatural state. Thus, a protein which is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be substantially free from itsnaturally-associated components. The term is used to describe receptorsand nucleic acids which have been synthesized in heterologous mammaliancells or plant cells, E. coli and other prokaryotes.

A polypeptide is substantially an entire membrane bound form of an FGF-Rwhen it is substantially a full length peptide corresponding to, orhighly homologous to a naturally occurring membrane bound form of anFGF-R.

Whether soluble or membrane bound, the present invention provides forsubstantially pure preparations. Various methods for their isolationfrom biological material may be devised, based in part upon thestructural and functional descriptions contained herein.

FGF receptor peptides including chicken and human FGF receptors may bepurified using techniques of classical protein chemistry, see below. Forexample, a lectin affinity chromatography step may be used, followed bya highly specific ligand affinity chromatography procedure that utilizesan FGF conjugated to biotin through the cysteine residues of the FGF.Purified FGF-R receptors may also be obtained by a method such as FGFaffinity chromatography using activated CH-Sepharose coupled to FGFthrough primary amino groups as described in Imamura, suDra. Thismethod, however, while resulting in a purified protein, may not providea workable amount of purified protein (i.e. more than nanogram amounts).

Depending on the availability of specific antibodies, as providedherein, specific FGF receptors may also be purified using immunoaffinitychromatography. Antibodies prepared, as described below, may beimmobilized to an inert substance to generate a highly specific affinitycolumn. See Harlow and Lane, below.

By way of example and not limitation, one purification procedure may beused which takes advantage of the fact that labeled biotin-bFGF bindswith high affinity to receptors in cells containing high amounts ofthose receptors. ¹²⁵I-labeled biotin-bFGF will bind to bFGF receptors inSwiss 3T3 cells and can be cross-linked to the receptor protein.

Various cell or tissue sources may be selected as starting materialsusually selected due to an abundance of the desired receptor. Chickenembryos (day 6, stage 29-30) are preferred because they containrelatively large amounts of the receptor protein as determined byhigh-affinity binding of human and bovine bFGF. Embryo extracts canfirst be fractionated on wheat germ agglutinin (WGA) Sepharose 4B andthe partially purified bFGF receptors then bound to biotin-bFGF. Thereceptor-ligand complex may be adsorbed to an avidin-agarose due to thehigh affinity interaction between the biotin and avidin moieties. Theavidin-agarose columns may be eluted with compounds which dissociate theFGF from its receptor such as suramin or SDS. The chicken protein whichbound to avidin-agarose in an FGF-dependent manner migrated at theexpected size (130 kDa) of the bFGF receptor. See FIG. 2B.

To determine the amino acid sequence or to obtain polypeptide fragmentsof the receptor, the receptor may be digested with trvpsin. Peptidefragments may be separated by reversed-phase high performance liquidchromatography (HPLC) and analyzed by gas-phase sequencing. Othersequencing methods known in the art may also be used.

The FGF receptors or the specific external regions of the receptors maybe used to affinity purify respective FGFS. The external regioncomprising the ligand-binding domain of the chicken bFGF-R shown in FIG.3 extends from about amino acid 22 to about amino acid 374. Theligand-binding domain of the human FGF-R shown in FIG. 4 extends fromabout amino acid 22 to about amino acid 285. The ligand-binding domainvaries with different FGF receptors and may be anywhere from 5% to 100%of the extracellular region. The minimal amount of protein sequencenecessary for ligand bonding may be determined by excising varioussegments of the extracellular domain and assaying ligand binding to theremaining sequence. Studies of ligand-receptor interaction indicate thatat least the ligand-binding region is located in the extracellularregion of the receptor is required. As used in this application, FGFreceptor or FGF-R ligand-binding activity means having the ability tobind a fibroblast growth factor or other specific ligand. Usually theseligands will be members of the FGF family. Therefore the external regionhas utility in establishing FGF agonists or antagonists.

It is also likely that the FGF-R, like many other growth factorreceptors, is found naturally in a multimeric protein complex, mostlikely in dimer form. Thus, other important regions of a receptor willbe those, either extracellular or otherwise, which are involved indimerization.

The intracellular regions of the receptors (e.g. starting at about aminoacid 396 through the COOH-terminus for the chicken bFGF-R and aboutamino acid 307 through the COOH-terminus for the human FGF-R shown inFIGS. 3 and 4, respectively) may also be used as enzymes with tyrosinekinase activity. The bek gene has 84% amino acid sequence identity tothe analogous region (tyrosine kinase region) of the chicken bFGF-R. Theflg has 99% homology with various sequences of the human FGF receptordescribed in FIG. 4.

A signal or leader sequence directs a protein through the membrane of acell. The signal sequences of the receptors may be used in conjunctionwith their respective receptors but may also be used with other proteins(e.g. amino acids about 1 through 21 of the N-terminal sequence comprisethe leader or signal sequence of the chicken bFGF-R shown in FIG. 3 andthe human FGF-R shown in FIG. 4).

The present invention also provides for analogues of the fibroblastgrowth factor receptor polypeptides. Such analogues include bothmodifications to a polypeptide backbone and variants and mutants of thepolypeptides. Modifications include chemical derivatizations ofpolypeptides, such as acetylations, carboxylations and the like. Theyalso include glycosylation modifications and processing variants of atypical polypeptide. These processing steps specifically includeenzymatic modifications, such as ubiquinization. See, e.g., Hershko andCiechanover (1982), “Mechanisms of Intracellular Protein Breakdown,”Ann. Rev. Bioch., 51:335-364.

Other analogues include genetic variants, both natural and induced.Induced mutants may be derived from various techniques including bothrandom mutagenesis of the encoding nucleic acids using irradiation orexposure to EMS, or may take the form of engineered changes bysite-specific mutagenesis or other techniques of modern molecularbiology. See, Sambrook, Fritsch and Maniatis (1989), Molecular Cloning:A Laboratory Manual (2d ed.), CSH Press.

Besides substantially full-length polypeptides, the present inventionprovides for biologically active fragments of the polypeptides.Significant biological activities include ligand-binding, immunologicalactivity and other biological activities characteristic of fibroblastgrowth factor receptor polypeptides. Immunological activities includeboth immunogenic function in a target immune system, as well as sharingof immunological epitopes for binding, serving as either a competitor orsubstitute antigen for a fibroblast growth factor receptor epitope. Asused herein, the term segment, as applied to a polypeptide, willordinarily be at least about 5 contiguous amino acids, typically atleast about 7 contiguous amino acids, more typically at least about 9contiguous amino acids, usually at least about 11 contiguous aminoacids, preferably at least about 13 contiguous amino acids, morepreferably at least about 16 contiguous amino acids, and most preferablyat least about 20 to 30 or more contiguous amino acids. Segments of aparticular domain will be segments of the appropriate size within thecorresponding domain.

For example, ligand-binding or other domains may be “swapped” betweendifferent new fusion polypeptides or fragments. Thus, new chimericpolypeptides exhibiting new combinations of specificities result fromthe functional linkage of ligand-binding specificities and intracellulardomains. For example, the Ig domains may be substituted by Ig domainsfrom other related polypeptides.

For immunological purposes, immunogens may be produced which tandemlyrepeat polypeptide segments, thereby producing highly antigenicproteins. Alternatively, such polypeptides will serve as highlyefficient competitors for specific binding. Production of antibodies tofibroblast growth factor receptor polypeptides is described below.

The present invention also provides for other polypeptides comprisingfragments of fibroblast growth factor receptors. Thus, fusionpolypeptides between the receptors and other homologous or heterologousproteins are provided. Homologous polypeptides may be fusions betweendifferent growth factor receptors, resulting in, for instance, a hybridprotein exhibiting ligand specificity of one receptor and theintracellular domain of another, or a receptor which may have broadenedor weakened specificity of binding. Likewise, heterologous fusions maybe constructed which would exhibit a combination of properties oractivities of the derivative proteins. Typical examples are fusions of areporter polypeptide, e.g., luciferase, with a domain of a receptor,e.g., a ligand-binding domain, so that the presence or location of adesired ligand may be easily determined. See, e.g., Dull et al., U.S.Pat. No. 4,859,609, which is hereby incorporated herein by reference.Other gene fusion partners include bacterial β-galactosidase, trpEProtein A, β-lactamase, alpha amylase, alcohol dehydrogenase and yeastalpha mating factor. See, e.g., Godowski et al. (1988), Science241:812-816; and Experimental section below.

Fusion proteins will typically be made by either recombinant nucleicacid methods or by synthetic polypeptide methods. Techniques for nucleicacid manipulation are described generally, for example, in Sambrook etal. (1989), Molecular Cloning: A Laboratory Manual (2d ed.), Vols. 1-3,Cold Spring Harbor Laboratory, which are incorporated herein byreference. Techniques for synthesis of polypeptides are described, forexample, in Merrifield, J. Amer. Chem. Soc. 85:2149-2156 (1963). Therecombinant nucleic acid sequences used to produce fusion proteins ofthe present invention may be derived from natural or syntheticsequences. Many natural gene sequences are obtainable from various cDNAor from genomic libraries using appropriate probes. See, GenBank™,National Institutes of Health. Typical probes for fibroblast growthfactor receptors may be selected from the sequences of FIGS. 3, 4, or 9in accordance with standard procedures. Suitable synthetic DNA fragmentsmay be prepared by the phosphoramidite method described by Beaucage andCarruthers, Tetra. Letts. 22:1859-1862 (1981). A double strandedfragment may then be obtained either by synthesizing the complementarystrand and annealing the strand together under appropriate conditions orby adding the complementary strand using DNA polymerase with anappropriate primer sequence.

III. NUCLEIC ACIDS

The present invention provides nucleic acid sequences encoding variousFGF receptor sequences described above. FIGS. 3, 4, and 7 respectivelyset forth the corresponding cDNA sequences encoding chicken and humanFGF receptors.

In FIG. 3 showing the chicken bFGF-R, peptides sequenced from purifiedprotein are underlined, including the NH₂-proximal sequences from aminoacids 35-3 (ala - - - arg), 56-67 (leu - - - arg), and 139-158(glu - - - lys). The transmembrane sequence is indicated by a dark bar,a unique acidic amino acid region is outlined, cysteine residues arecircled, potential N-linked glycosylation sites are indicated by a dotand the dashed underlining indicates the putative hydrophobic signalsequence. The amino acid sequence includes an in-frame stop codon (aboutresidue −12) followed by an initiator methionine. The structuralsequence begins at about amino acid 22.

In FIG. 4 showing the human FGF-R, the methionine of codon ATG startingat about nucleotide 529 is the first amino acid of the FGF-R gene. Forexample, amino acid 22 of the receptor described in FIG. 4 is anarginine residue (R) located two amino acids in from the left, two linesup from the bottom between “589” and “630” on page 1 of FIG. 4.

Nucleic acids according to the present invention will possess a sequencewhich is either derived from a natural human, chicken, or other FGF-Rgene or one having substantial homology with a natural FGF-R gene or aportion thereof.

Substantial homology in the nucleic acid context means either that thesegments, or their complementary strands, when optimally aligned andcompared, are identical with appropriate nucleotide insertions ordeletions, in at least about 80% of the residues, usually at least about90%, more usually at least about 95%, preferably at least about 97%, andmore preferably at least about 98 to 99.5% of the nucleotides.Alternatively, substantial homology exists when the segments willhybridize under selective hybridization conditions, to a strand, or itscomplement, typically using a sequence derived from FIGS. 3, 4, or 9.Selectivity of hybridization exists when hybridization occurs which ismore selective than total lack of specificity. Typically, selectivehybridization will occur when there is at least about 55% homology overa stretch of at least about {fraction (14/25)} nucleotides, preferablyat least about 65%, more preferably at least about 75%, and mostpreferably at least about 90%. See, Kanehisa, M. (1984), Nucleic AcidsRes. 12:203-213, which is incorporated herein by reference. Stringenthybridization conditions will typically include salt concentrations ofless than about 1 M, more usually less than about 500 mM and preferablyless than about 200 mM. Temperature conditions will typically be greaterthan 20° C., more usually greater than about 30° C. and preferably inexcess of about 37° C. As other factors may significantly affect thestringency of hybridization, including, among others, base compositionand size of the complementary strands, presence of organic solvents andextent of base mismatching, the combination of parameters is moreimportant than the absolute measure of any one.

An isolated nucleic acid is one which has been substantially purifiedaway from other sequences which normally accompany it, e.g., othercellular nucleic acid sequences. Usually, the term refers to a fragmentof a genome which has been selectively cloned, isolated and purified tosubstantial homogeneity.

Probes may be prepared based on the sequence of the FGF receptor cDNAsprovided in FIGS. 3, 4, and 9. The probes will include an isolatednucleic acid attached to a label or reporter molecule and may be used toisolate other FGF receptor nucleic acid sequences by standard methods.See, e.g. J. Sambrook et al., Molecular Cloning: A Laboratory Manual,vols. 1-3, CSH Press, N.Y. (1989), which is hereby incorporated hereinby reference. Other similar nucleic acids may be selected for by usinghomologous nucleic acids. Alternatively, nucleic acids encoding thesesame or similar receptor polypeptides may be synthesized or selected bymaking use of the redundancy in the genetic code. Various codonsubstitutions may be introduced, e.g., silent changes thereby producingvarious restriction sites, or to optimize expression for a particularsystem. Mutations may be introduced to modify the properties of thereceptors, perhaps to change the ligand binding affinities, theinter-chain affinities, or the polypeptide degradation or turnover rate.

The DNA compositions of this invention may be derived from genomic DNAor cDNA, prepared by synthesis or may be a hybrid of the variouscombinations. Recombinant nucleic acids comprising sequences otherwisenot naturally occurring are also provided by this invention. An isolatedDNA sequence includes any sequence that has been obtained by primer orhybridization reactions or subjected to treatment with restrictionenzymes or the like.

Synthetic oligonucleotides can be formulated by the triester methodaccording to Matteucci, et al., J. Am. Chem. Soc., 103:3185 (1981) or byother methods such as commercial automated oligonucleotide synthesizers.Oligonucleotides can be labeled by excess polynucleotide kinase (e.g.,about 10 units to 0.1 nmole substrate is used in connection with 50 mMTris, pH 7.6, 5 mM dithiothreitol, 10 mM MgCl₂, 1-2 mM ATP, 1.7 pmoles³²P-ATP (2.9 mCi/mmole) 0.1 mM spermidine, 0.1 mM EDTA). Probes may alsobe prepared by nick translation, Klenow fill-in reaction, or othermethods known in the art.

cDNA or genomic libraries of various types may be screened. The choiceof cDNA libraries normally corresponds to a tissue source which isabundant in mRNA for the desired receptors. Phage libraries are normallypreferred, but plasmid libraries may also be used. For example, akeratinocyte cell genomic or cDNA library would be preferred to isolateand clone a keratinocyte growth factor receptor. Embryonic or placentallibraries can be used for int-2, FGF-5 and hst receptors and anendothelial cell library is preferred for acidic FGF receptors. Clonesof a library are spread onto plates, transferred to a substrate forscreening, denatured and probed for the presence of desired sequences.

For example, with a plaque hybridization procedure, each platecontaining bacteriophage plaques is replicated onto duplicatenitrocellulose filter papers (Millipore-HATF). The phage DNA isdenatured with a buffer such as 500 mM NaOH, 1.5 M NaCl for about 1minute, and neutralized with, e.g., 0.5 M Tris-HCl, pH 7.5, 1.5 M NaCl(3 times for 10 minutes each). The filters are then washed. Afterdrying, the filters are typically baked, e.g., for 2 hours at 80° C. ina vacuum oven. The duplicate filters are prehybridized at 42° C. for4-24 hours with 10 ml per filter of DNA hybridization buffer (20-50%formamide, 5×SSC, pH 7.0, 5×Denhardt's solution (polyvinylpyrrolidone,plus Ficoll and bovine serum albumin; 1X=0.02% of each), 50 mM sodiumphosphate buffer at pH 7.0, 0.2% SDS, and 50 μg/ml denatured salmonsperm DNA). Hybridization with an appropriate probe may be performed at42° C. for 16 hrs with 10 ml/filter of 1×10⁶ cpm/ml of DNA hybridizationbuffer containing labeled probe. The final concentration of formamide isvaried according to the length of the probe and the degree of stringencydesired. See, e.g., J. G. Wetmur ad Davidson, J. Mol. Biol. 31:349-370(1968); and M. Kanehisa, Nuc. Acids Res. 12:203-213 (1984), each ofwhich is incorporated herein by reference, for a discussion ofhybridization conditions and sequence homology.

An oligonucleotide probe based on the amino acid sequence of the twotryptic peptides of the purified chicken bFGF-R was used to screen achicken embryo (day 6) cDNA library under low stringency conditions.Sequences corresponding to TVALGSNVEFVCK and VYSDPQPHIQWLY, preparedusing a commercial automated oligonucleotide synthesizer (AppliedBiosystems) were used to obtain the chicken bFGF receptor clonedescribed in FIG. 3. This clone, or sequences derived from it, can beused to isolate bFGF-Rs in other species as well as other FGF-Rs in atarget species.

The probes described above which were used to isolate the chicken bFGF-Rwere also used to isolate a human bFGF receptor CDNA clone.

In accordance with this invention any isolated DNA sequence whichencodes an FGF-R complete structural sequence can be used as a probe.Alternatively, any DNA sequence that encodes an FGF-R hydrophobic signalsequence and its translational start site may be used. Any isolatedpartial DNA sequence which encodes an FGF-R activity (e.g.ligand-binding or FGF-R binding) is also part of this invention.Preferred probes are cDNA clones of each isolated FGF receptor.

The DNA sequences used in this invention will usually comprise at leastabout 5 codons (15 nucleotides), more usually at least about 7 codons,typically at least about 10 codons, preferably at least about 15 codons,more preferably at least about 25 codons and most preferably at leastabout 35 codons. One or more introns may also be present. This number ofnucleotides is usually about the minimal lenath required for asuccessful probe that would hybridize specifically with an FGF receptor.For example, epitopes characteristic of an FGF-R may be encoded in shortpeptides. Usually the wild-type sequence will be employed, in someinstances one or more mutations may be introduced, such as deletions,substitutions, insertions or inversions resulting in changes in theamino acid sequence to provide silent mutations, to modify a restrictionsite, or to provide specific mutations. The genomic sequence willusually not exceed about 200 kb, more usually not exceed about 100 kb,preferably not be greater than 0.5 kb.

Portions of the DNA sequence having at least about 15 nucleotides,usually at least about 15 nucleotides, and fewer than about 6 kd,usually fewer than about 1.0 kb, from a DNA sequence encoding an FGFreceptor are preferred as probes. The probes may also be used todetermine whether mRNA encoding a specific FGF-R is present in a cell ordifferent tissues.

The natural or synthetic DNA fragments coding for a desired fibroblastgrowth factor receptor fragment will be incorporated into DNA constructscapable of introduction to and expression in an in vitro cell culture.Usually the DNA constructs will be suitable for replication in aunicellular host, such as yeast or bacteria, but may also be intendedfor introduction to, with and without and integration within the genome,cultured mammalian or plant or other eukaryotic cell lines. DNAconstructs prepared for introduction into bacteria or yeast willtypically include a replication system recognized by the host, theintended DNA fragment encoding the desired receptor polypeptide,transcription and translational initiation regulatory sequences operablylinked to the polypeptide encoding segment and transcriptional andtranslational termination regulatory sequences operably linked to thepolypeptide encoding segment. The transcriptional regulatory sequenceswill typically include a heterologous enhancer or promoter which isrecognized by the host. The selection of an appropriate promoter willdepend upon the host, but promoters such as the trp, lac and phagepromoters, tRNA promoters and glycolytic enzyme promoters are known.See, Sambrook et al. (1989). Conveniently available expression vectorswhich include the replication system and transcriptional andtranslational regulatory sequences together with the insertion site forthe fibroblast growth factor receptor DNA sequence may be employed.Examples of workable combinations of cell lines and expression vectorsare described in Sambrook et al. (1989); see also, Metzger et al.(1988), Nature 334:31-36.

Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter, an enhancer andnecessary processing information sites, such as ribosome-binding sites,RNA splice sites, polyadenylation sites, and transcriptional terminatorsequences. Preferably, the enhancers or promoters will be thosenaturally associated with genes encoding the fibroblast growth factorreceptors, although it will be understood that in many cases others willbe equally or more appropriate. Other preferred expression controlsequences are enhancers or promoters derived from viruses, such as SV40,Adenovirus, Bovine Papilloma Virus, and the like.

Similarly, preferred promoters are those found naturally inimmunoglobulin-producing cells (see, U.S. Pat. No. 4,663,281, which isincorporated herein by reference), but SV40, polyoma virus,cytomegalovirus (human or murine) and the LTR from various retroviruses(such as murine leukemia virus, murine or Rous sarcoma virus and HIV)may be utilized, as well sa promoters endogenous to FGF-R genes. See,Enhancers and Eukarvotic Gene Expression, Cold Spring Harbor Press,N.Y., 1983, which is incorporated herein by reference.

The vectors containing the DNA segments of interest (e.g., a fibroblastgrowth factor receptor gene or cDNA sequence or portions thereof) can betransferred into the host cell by well-known methods, which varydepending on the type of cellular host. For example, calcium chloridetransfection is commonly utilized for procaryotic cells, whereas calciumphosphate treatment may be used for other cellular hosts. See generally,Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual (2d ed.),CSH Press (1989), which is incorporated herein by reference. The term“transformed cell” is meant to also include the progeny of a transformedcell.

As with the purified polypeptides, the nucleic acid segments associatedwith the ligand-binding segment, the extracellular domain and theintracellular domain are particularly useful. These gene segments willbe used as probes for screening for new genes exhibiting similarbiological activities, though the controlling elements of these genesmay also be of importance.

IV. METHODS FOR MAKING FGF RECEPTORS

DNA sequences may also be used to express polypeptides which exhibit orinhibit FGF receptor activity. For example, a DNA sequence of from about21 nucleotides (about 7 amino acids) to about 2.1 kb (about 700 aminoacids) may be used to express a polypeptide having an FGF receptorspecific activity, typically ligand-binding.

Large quantities of the receptor proteins may be prepared by expressingthe whole receptor or parts of the receptor contained in the expressionvehicles in compatible hosts such as E. coli, yeast, mammalian cells,insect cells or frog oocytes. The expression vehicles may be introducedinto the cells using methods well known in the art such as calciumphosphate precipitation (discussed below), lipofection, electroporationor DEAE dextran.

Usually the mammalian cell hosts will be immortalized cell lines. Tostudy the characteristics of an FGF-R and its corresponding growthfactor, it will be useful to transfect, etc. mammalian cells which lackor have low levels of an FGF receptor where the signal sequence directsthe peptide into the cell membrane. Cells without significant FGFreceptors include lymphocytes, myocytes, green monkey cos-7 cells andChinese hamster ovary cells (CHO). Transformed or transfected, etc.,cells encode a receptor that is functionally equivalent to a wild-typereceptor and confers a FGF-sensitive mitogenic response on the cell.Such cells will enable one to analyze the binding properties of variousnative FGFs. Transfected cells may also be used to evaluate acomposition or drug's effectiveness as an FGF antagonist or agonist. Thelevel of receptor tyrosine kinase activity or the rate of nucleic acidsynthesis can be determined by contacting transfected cells with drugsand comparing the effects of FGFs or their analogs on the drug-treatedcells versus the controls. Although the most common prokaryote cellsused as hosts are strains of E. coli, other prokaryotes such as Bacillussubtilis or Pseudomonas may also be used. The DNA sequence of theinvention, including fragments or portions of the sequence encoding foran entire receptor, a portion of the receptor or a polypeptide having anFGF-R activity can be used to prepare an expression vehicle or constructfor an FGF-R or polypeptide having an FGF-R activity. Usually thecontrol sequence will be a eukaryotic promoter for expression in amammalian cell. In some vehicles, the receptor's own control sequencesmay also be used. A common procaryotic plasmid vector for transformingE. coli is pBR322 or its derivatives (e.g. the plasmid pkt279(Clontech)) (Bolavar et al., Gene, 2:95 (1977)). The procaryotic vectorsmay also contain procaryotic promoters for transcription initiation,optionally with an operator. Examples of most commonly used procaryoticpromoters include the beta-lactamase (penicillinase) and lactose (lac)promoter (Cheng et al., Nature, 198:1056 (1977), the tryptophan promoter(trp) (Goeddell et al., Nucleic Acid Res., 8: 457 (1980)) the P_(L)promoter and the N-gene ribosome binding site (Shimatake et al., Nature,292:128 (1981).

Promoters used in conjunction with yeast can be promoters derived fromthe enolase gene (Holland et al., J Biol. Chem., 256:1385 (1981)) or thepromoter for the synthesis of glycolytic enzymes such as3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255 (1980)).

Appropriate non-native mammalian promoters might include the early andlate promoters from SV40 (Fiers et al., Nature, 273:113 (1978) orpromoters derived from murine molony leukemia virus, mouse mammary tumorvirus, avian sarcoma viruses, adenovirus II, bovine papilloma virus orpolyoma. In addition, the construct may be joined to an amplifiable gene(e.g. DHFR) so that multiple copies of the FGF receptor gene may bemade.

Prokaryotes may be transformed by various methods, including using CaCl₂(Cohen, S. N., Proc. Natl. Acad. Sci. USA, 69:2110 (1972)) or the RbClmethod (Maniatis et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press 1982)). Yeast may be transformed using a methoddescribed by Van Solingen et al., J. Bacter., 130:946 (1977) and C. L.Hsiao et al., Proc. Natl. Acad. Sci. USA, 76:3829 (1979). With respectto eukaryotes, mammalian cells may be transfected using a calciumphosphate precipitation method described by (Graham and van der Eb,Virology, 52:546 (1978)), or by lipofectin (BRL) or retroviral infection(E. Gilboa, Experimental Manipulation of Gene Expression, Chap. 9,Academic Press P. 175 (1983)). The actual expression vectors containingappropriate sequences may be prepared according to standard techniquesinvolving ligation and restriction enzymes (See e.g., Maniatis supra.)Commercially available restriction enzymes for cleaving specific sitesof DNA may be obtained from New England BioLabs, Waltham, Mass.

Clones are selected by using markers depending on the mode of the vectorconstruction. The marker may be on the same or a different DNA moleculepreferably the same DNA molecule. With mammalian cells the receptor geneitself may be the best marker. In procaryotic hosts the transformant maybe selected by resistance to ampicillin, tetracycline or otherantibiotics. Production of a particular product based on temperaturesensitivity may also serve as an appropriate marker. Various methods maybe used to harvest and purify the FGF-R receptor protein or peptidefragment. The peptide may be isolated from a lysate of the host. Thepeptide may be isolated from the cell supernatant if the peptide issecreted. The FGF-R peptide is then further purified as discussed aboveusing HPLC, electrophoresis, affinity chromatography (preferablyimmuno-affinity or ligand affinity).

Another method which can be used to isolate cDNA clones of FGF-R relatedspecies involves the use of the polymerase chain reaction (PCR). (Saiki,R. K., et al. Science 230: 1350 (1985). In this approach twooligonucleorides (27mers) corresponding to distinct regions of the FGF-Rsequence are synthesized and then used in the PCR reaction to amplifyreceptor-related mRNA transcripts from an mRNA source. Annealing of theoligonucleotides and PCR reaction condition are performed underconditions of reduced stringency as described below in Example 2. Theresulting amplified fragments are subcloned, and the resultingrecombinant colonies are probed with ³²P-labeled full-length FGF-R CDNAusing both high and low stringency conditions (see Examples 2 and 3).Clones which hybridize under low but not high stringency conditionsrepresent FGF-R related mRNA transcripts. In addition this approach canbe used to isolate variant FGF-R cDNA species which arise as a result ofalternative splicing, see Frohman, M. A., et al., Proc. Natl. Acad. Sci.USA, 85: 8998 (1988).

V. ANTIBODIES

Polyclonal and/or monoclonal antibodies to the various FGF receptors andpeptide fragments may also be prepared. The term antibody is used bothto refer to a homogeneous molecular entity, or a mixture such as a serumproduct made up of a plurality of different molecular entities. Peptidefragments may be prepared synthetically in a peptide synthesizer andcoupled to a carrier molecule (i.e. keyhole limpet hemocyanin) andinjected into rabbits over several months. The rabbit sera is tested forimmunoreactivity to the FGF receptor protein or fragment. Monoclonalantibodies may be made by injecting mice with FGF-R protein, FGF-Rpolypeptides or mouse cells expressing high levels of the cloned FGFreceptor on its cell surface. Monoclonal antibodies will be screened byELISA and tested for specific immunoreactivity with the FGF receptorprotein or polypeptides thereof. See, E. Harlow and D. Lane, Antibodies:A Laboratory Manual, CSH Laboratories (1988), which is herebyincorporated herein by reference. These antibodies will be useful inassays as well as pharmaceuticals.

Once a sufficient quantity of the desired fibroblast growth factorreceptor polypeptide has been obtained, the protein may be used forvarious purposes. A typical use is the production of antibodies specificfor binding to these receptors. These antibodies may be eitherpolyclonal or monoclonal and may be produced by in vitro or in vivotechniques.

For production of polyclonal antibodies, an appropriate target immunesystem is selected, typically a mouse or rabbit. The substantiallypurified antigen is presented to the immune system in a fashiondetermined by methods appropriate for the animal and other parameterswell known to immunologists. Typical sites for injection are in thefootpads, intramuscularly, intraperitoneally, or intradermally. Ofcourse, another species may be substituted for a mouse or rabbit.

An immunological response is usually assayed with an immunoassay.Normally such immunoassays involve some purification of a source ofantigen, for example, produced by the same cells and in the same fashionas the antigen was produced. The immunoassay may be a radioimmunoassay,an enzyme-linked assay (ELISA), a fluorescent assay, or any of manyother choices, most of which are functionally equivalent but may exhibitadvantages under specific conditions.

Monoclonal antibodies with affinities of 10⁸ M⁻¹ preferably 10⁹ to 10¹⁰,or stronger will typically be made by standard procedures as described,e.g., in Harlow and Lane, Antibodies: A Laboratory Manual, CSHLaboratory (1988); or Goding, Monoclonal Antibodies: Principles andPractice (2d ed) Academic Press, New York (1986), which are herebyincorporated herein by reference. Briefly, appropriate animals will beselected and the desired immunization protocol followed. After theappropriate period of time, the spleens of such animals are excised andindividual spleen cells fused, typically, to immortalized myeloma cellsunder appropriate selection conditions. Thereafter the cells areclonally separated and the supernatants of each clone are tested fortheir production of an appropriate antibody specific for the desiredregion of the antigen.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides or alternatively to selection of libraries ofantibodies in phage or similar vectors. See, Huse et al., “Generation ofa Large Combinatorial Library of the Immunoglobulin Repertoire in PhageLambda,” Science 246:1275-1281 (1989), hereby incorporated herein byreference. The polypeptides and antibodies of the present invention maybe used with or without modification. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent agents, chemiluminescent agents, magneticparticles and the like. Patents, teaching the use of such labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241. Also, recombinant immunoglobulins may beproduced, see Cabilly, U.S. Pat. No. 4,816,567.

VIII. METHODS FOR USE

The present invention provides a fibroblast growth factor-receptor(FGF-R) purification method as well as a method for synthesizing FGFreceptors within cells. Also provided are the homogeneous receptorsproduced by these methods, the nucleic acid sequences encoding thereceptors or portions of the receptors, as well as the expressionvehicles containing these sequences, cells comprising the FGF-receptorsand antibodies to the receptors. Of particular interest are the solubleforms of the receptors, which have binding sites which may compete withreceptors to bind FGF.

However, as indicated above, the FGF-R likely functions in a dimerstate. The soluble forms of the receptor may interfere with thedimerization and may be effective in blocking signal transduction by adifferent mechanism from competitive affinity for the FGF ligands. Thesoluble, or intracellular or transmembrane fragments of the variousreceptor forms are expected to interfere with dimer formation and thuscan serve to block at least some types of, or some fraction of signaltransduction.

This observation provides a method for modifying in vivo a fibroblastgrowth factor receptor modulated activity comprising administering to apatient an amount of a fibroblast growth factor receptor blocking agenteffective to inhibit fibroblast growth factor binding to fibroblastgrowth factor receptors. As discussed above, the FGF family of proteinshave a significant role in regulating many important physiologicalprocesses. The soluble FGF-R polypeptides may be effective in modifyingthe extent of FGF modulation of these processes. For this reason, thesoluble forms of the receptors may find use as competitive binding sitesfor FGF. Likewise, truncated FGF binding sites or binding sites whichhave been mutated, particularly those from the human forms described,may be equally effective in this effect at a lesser cost, both in termsof economics and in terms of medical side-effects upon administration.

The reagents provided herein will also find use in diagnosis of eitherFGF production or FGF-R production. Various medical conditions areindicated by an abnormal level of production of either of theseproteins, including, e.g., Kaposi sarcoma, which produces Kaposi FGF,and diabetic retinopathy. Thus, diagnostic tests dependent upon thesereagents now become available.

With the different FGF types, there is a likelihood that different typesof receptors exist having variations in affinities for the variousligands. With the genes and proteins of the present invention,distinctions between various receptor types will be found. Thus, tissuemarkers should become available.

Since tumor growth is so dependent upon microvascularization,administration of the FGF-R may serve to prevent such and result insuppression of tumor growth. By prevention of the FGF activation, thepresent invention may be an important addition to the arsenal of agentsfor fighting tumor growth.

Viral infections may also be dependent upon binding to particularreceptors for the invasion process. There is suggestive evidence thatHSV (Herpes simplex virus) infects by binding to FGF-R proteins. Thus,administration of therapeutically effective amounts of FGF-R solubleforms or fragments may serve as a prophylactic measure to minimize therisk of exposure to this, or other viruses, making use of this mechanismfor cell entry. Again, the mechanism of protection may depend uponcompetitive binding, disruption of dimer structure, a combination, oranother.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicantsadministered. Thus, one should titrate the dosage for treatment ofparticular conditions. Typically, dosages used in vitro may provideuseful guidance in the amounts useful for in situ administration ofthese reagents. Animal testing of dosages for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described in Gilman et al., Goodman andGilman's: The Pharmacological Basis of Therapeutics, 7th Ed., MacMillan,New York (1985), which is hereby incorporated herein by reference.Because of the high affinity binding between FGF and its receptors, lowdosages of these reagents would be initially expected to be effective.Thus, dosage ranges would ordinarily be expected to be in amounts lowerthan mM concentrations, typically less than about 10 μM concentrations,usually less than about 100 nM concentrations, more usually less thanabout 1 nM, preferably less than about 10 pM (picomolar), morepreferably less than about 100 fM (femtomolar), and most 30 preferablyless than about 1 fM, with an appropriate carrier.

The invention will better be understood by reference to the followingillustrative examples.

EXAMPLE 1 Characterization of a bFGF Receptor

¹²⁵I-labeled bFGF was first competitively bound to Swiss 3T3 cells. Asshown in FIG. 1(A), ¹²⁵I-labeled bFGF (2 Ci/μmol) was added to theconfluent 3T3 cells (6 fmol of ¹²⁵I-labeled bFGF per 10⁵ cells) in thepresence of indicated concentrations of: unmodified bFGF (-X-);biotin-bFGF (solid square); the unbound fraction after biotin-bFGF wasincubated with avidin-agarose, (open square); the unbound fraction afterbFGF was incubated with avidin-agarose, (open triangle). Binding wasperformed for 30 min at 37° C. in culture media (DME H21) containing0.2% gelatin, and heparin (15 U/ml). The cells were washed three timeswith a buffer containing 20 mM HEPES (pH 7.4), 0.2% gelatin, and 150 mMNaCl. The radioactivity present was determined in a Beckman gammacounter. Maximal binding (0% inhibition) represents 5700 cpm of specificbinding (nonspecific binding was 600 cpm). All determinations were madein triplicate. Recombinant human bFGF (Barr et al., J. Biol. Chem., 263:16471 (1988)) was iodinated using IODOBEADS (Pierce). The bFGF wasiodinated using 0.5- lmCi; of ¹²⁵I per 1 μg FGF, 0.2M NaPi, pH 7.4, 2IODOBEADS and incubated for 15 min. at room temperature, quenched withNa metabisulfite and excess KI. Iodinated bFGF was separated fromunreacted free iodine by gel filtration on a PD 10 column equilibratedwith 0.2M Na phosphate, pH 7.5, 0.2M NaCl, 0.2% gelatin. The bFGF wasbiotinylated using iodoacetyl-LC-biotin (Pierce) at a 4:1 molar excessof cysteine residues in 10 mM Tris-HCl (pH 8.0) for 5 hours at 4° C.,according to the method of Yamamoto, et al., FEBS Lett. 176:75 (1984).Unreacted biotin was removed by gel filtration with PD 10 columns asdescribed above (Pharmacia). During the purification procedure, modifiedbFGF was indistinguishable from unmodified bFGF in its ability toinhibit the binding of ¹²⁵I-labeled bFGF to high affinity bFGF receptorsin Swiss 3T3 cells and in its ability to stimulate the phosphorylationof a 90 kD protein, known to be a substrate of bFGF-induced tyrosinekinase activity. See FIG. 1(A). The biotinylation reaction modified 90to 95% of the bFGF molecules as measured by binding to avidin-conjugatedagarose.

As shown in FIG. 1(B), cellular in situ bFGF receptors were cross-linkedto labeled bFGF. ¹²⁵I-labeled biotin-bFGF or 125I-labeled bFGF (0.1pmol) was added to Swiss 3T3 cells (5×10⁵ cells) in the presence orabsence of unlabeled bFGF as indicated. The cells were washed andcross-linked with 0.15 mM disuccinimidyl suberate (DSS) (Pierce). Thecells were then solubilized, subjected to SDS polyacrylamide gelelectrophoresis (PAGE) and ¹²⁵I-labeled proteins were detected byautoradiography. ¹²⁵I-labeled biotin-bFGF bound to bFGF receptors inSwiss 3T3 cells with high affinity (dissociation constant equals 1 nM)and was cross-linked to a 130 kD protein which comigrated with the bFGFreceptor cross-linked to ¹²⁵I-labeled bFGF.

Purified chicken bFGF receptor was prepared by homogenizing fresh day 6chicken embryos (stage 29-30) with a Brinkmann polytron; (1500embryos/batch); (1:1 v/v) in a final concentration of 0.25 M sucrose, 50mM HEPES (pH 7.5), 2 mM EDTA, 50 mM NaF, 150 μM sodium orthovanadate, 30mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF),aprotinin (20 to 30 kallikrein international units (KIU)/ml, leupeptin(10 μg/ml), and pepstatin (1 μg/ml). The homogenate was centrifuged at17,700 g for 45 minutes at 4° C. The pellet was resuspended inhomogenization buffer (300 ml) and the resulting suspension was referredto as the membrane fraction (Mb). The membrane fraction was thenincubated for 30 min at 4° C. with an equal volume of 2×lysis buffer(1×lysis buffer consists of 10 mM Tris-HCl (pH 7.5)), 50 mM NaCl, 5 mMEDTA, 1% 25 Triton X-100, 50 mM NaF, 150 μM sodium orthovanadate, 30 mMsodium pyrophosphate, 1 mM PMSF, aprotinin (20 to 30 KIU/ml), leupeptin(10 μg/ml) and pepstatin (1 μg/ml)), and then centrifuged at 31,000 gfor 30 min. The supernatant was applied batchwise to a 150 mlWGA-Sepharose 4B column, washed with 300 ml of lysis buffer followed by500 ml of column buffer which contained 20 mM HEPES (pH 7.5), 2 mM EDTA,10% glycerol, 0.1% Triton X-100, 50 mM NaF, 150 μM sodium orthovanadate,30 mM sodium pyrophosphate, 1 mM PMSF, aprotinin (20 to 30 KIU/ml),leupeptin (10 μg/ml) and pepstatin (1 μg/ml). The column was eluted withcolumn buffer containing 0.5 M N-acetylglucosamine. Peak proteincontaining fractions were combined and stored at −70° C.

To establish the presence of FGF-R in the embryo membranes and WGAeluate, chicken bFGF receptor was cross-linked by incubating 10 μl ofthe chicken embryo membrane fraction (Mb) or 100 μl of the eluate fromthe WGA-Sepharose 4B column with ¹²⁵I-labeled bFGF (0.1 pmol) in thepresence (+) or absence (−) of a 200-fold excess of unlabeled BFGF for30 min at 37 C. (See FIG. 2(A)). DSS was added to a concentration of0.15 mM, and the reaction mixture was incubated for 10 min on ice.Samples were subjected to SDS PAGE followed by autoradiography. Specificbinding and cross-linking of ¹²⁵I-bFGF to crude chicken embryo membranefraction revealed only a single protein band of 150 kDa (FIG. 2(A)).After the molecular mass of bFGF was subtracted, the deduced size of thechicken bFGF receptor was 130-135 kDa.

As shown in FIG. 2(B), two large-scale ligand affinity purificationswere performed (each using the material from 20,000 embryos). The eluatefrom the WGA-Sepharose 4B column was incubated with biotin-bFGF preparedas described above (10:1 molar excess of ligand to receptor) and heparinat a concentration of 15 U/ml (to reduce low affinity binding) for 30min at 4° C. The mixture was then cycled twice through a 10 mlavidin-agarose column (bFGF-agarose). To determine the nonspecificbinding of protein to avidin-agarose (control), the eluate from theWGA-Sepharose 4B column was cycled through avidin-agarose in the absenceof biotin-bFGF (control). The columns were washed with 200 ml of columnbuffer used with the Sepharose column described above containing 0.2 MNaCl followed by column buffer without NaCl (300 ml) and then elutedwith 10 mM suramin in column buffer. Four sequential 10 ml fractionswere collected (frac. 1-4) and samples of each fraction were subjectedto SDS PAGE and stained with silver nitrate. As shown in FIG. 2(B), onlya single protein bound to avidin-agarose in an FGF-dependent manner andit migrated at the expected size (130 kDa) of the bFGF receptor.

The eluted proteins were separated by acrylamide gel electrophoresis andstained with Coomassie Blue. The band corresponding to the bFGF receptorwas cut out and the protein electroeluted according to the method of M.W. Hunkapiller, et al., Meth. In Enzymol., 91: 227 (1983). Thisprocedure resulted in the purification of 2 to 5 ng of pure FGF receptorper chicken embryo with an overall recovery of 5%.

To further characterize the receptor, protein was digested with trypsin.Peptide fragments were isolated by reversed-phase high performanceliquid chromatography (HPLC) and analyzed by gas-phase sequencing asdescribed in Yarden et al., supra. From the two independentpreparations, the amino acid sequences of 14 peptides, as shown in FIG.3, were obtained. Three of the peptides were common to both preparationsindicating identity between the two independent isolations. Four of thetryptic peptides (LILGKPLGEGCFGQVVLA, IADFGLAR, MAPEALFDR andIYTHQSDVWSFGV, See Table I and FIG. 3) were homologous to consensussequences for tyrosine kinase domains (FIG. 6). This was consistent withthe finding that tyrosine kinase activity is associated with the bFGFreceptor as described in Huang and Huang, J. Biol. Chem. 261:9568(1986). Thus, the purified protein was determined to be a purified bFGFreceptor in that it bound to bFGF, was the expected molecular weight ofthe receptor, and contained tyrosine kinase sequences.

As discussed above the amino acid sequences of 11 of the 14 peptideswere identified in a previously published sequence of a partial humancDNA clone, termed flg (fms-like gene). See M. Ruta et al., Oncogene, 3:9 (1988). That sequence was isolated on the basis of its homology to theproto-oncogene sequence and was not previously recognized to encode atransmembrane receptor protein.

EXAMPLE 2 Isolation of a Full-Length Chicken bFGF Receptor cDNA Clone

A chicken embryo (day 6) cDNA library was constructed from size-selectedpoly A⁺mRNA. 200 μg of poly A⁺mRNA was size-fractionated on a 10%-30%sucrose gradient and fractions containing mRNA greater than or equal to3.5 Kb were pooled. 5 μg of the sized mRNA was used to generate the cDNAaccording to the method of U. Gubler and B. Hoffman, Gene 25:263 (1983)using a cDNA synthesis kit from Pharmacia (cat.#27-9260-01). Thesynthesized cDNAs were size-selected for cDNAs greater than or equal to2.0 kb, and the sized cDNAs were then cloned into the Eco RI site of thebacteriophage vector ZapII (Stratagene, cat.#236211). The resultant cDNAlibrary contained 2.0×10⁶ independent recombinants.

The library was screened with a ³²P-labeled oligonucleotide probe thatencoded the two contiguous peptides shown in FIG. 3 (TVALGSNVEFVCK andVYSDPQPHIQWLK). The oligonucleotides were prepared using a commercialautomated oligonucleotide synthesizer. Two 43-45 base oligonucleotidescontaining a 12 base overlapping complementary sequence were annealedand labeled by Klenow fill-in with dNTP's (-dCTP), ³²P-dCTP, and DNApolymerase Klenow fragment yielding a 70 bp labeled probe. Filters werehybridized under low stringency conditions (20% formamide, 5×standardsaline citrate (SSC) and 5×Denhardt's solution at 42° C.) and washedwith 0.2×SSC at 42° C. Twenty-five positive clones were isolatedfollowing 3 rounds of plaque purification. Of the 25 positive clones, 11hybridized at high stringency to the human FGF-R cDNA labeled by nicktranslation and used as a probe (see Example 3). All of the 11 cloneswere essentially identical except for variation in length at the 5′ endof the clones. The amino acid sequence of the largest clone (3.2 kb)contained the sequence of all 14 of the receptor peptides obtained inthe protein purification described above (See FIG. 3) and contained thecomplete coding sequence of the FGF-R. The transmembrane region and thehydrophobic signal sequence were identified by Kyte and Doolittlehydropathy analysis as described in Kyte and Doolittle, J. Mol. Biol.,157:105 (1982).

A single hybridizing band of approximately 3.5 Kb was identified byprobing chicken embryo poly(A)⁺RNA (5 μg) with full-length chicken bFGFreceptor cDNA under high stringency conditions (50% formamide),5×Denhardt's solution and 5×SSC at 42° C. Filters were then washed with0.2×SSC at 65°. The 3.5 kb single hybridizing band identified by the RNAblot analysis is shown in FIG. 5(A). Primer extension experiments withan oligonucleotide complementary to a sequence near the 5′ end of theclone were performed. Chicken embryo poly(A)⁺RNA (5 μg) was denaturedwith 10 mM methylmercury, annealed to ³²P-labeled primer (5′CTGCACGTCATCGCGCA-3′) and extended with murine Moloney leukemia virusreverse transcriptase. (See FIG. 5(B): lane (S) represents ³²P-labeledDNA molecular size standards (1 kb); Lane (E) represents extendedfragment (523 nucleotides); Lanes (G, A, T, and C) represent a 5%acrylamide sequencing gel. The data predicted that the mRNA of thereceptor was 48 nucleotides longer than the isolated clone.

The amino acid sequence of the longest open reading frame (2.4 kb)included an in-frame stop codon (amino acid residue −12) followed by aninitiator methionine (residue 1) and the entire receptor coding sequence(FIG. 3). The cDNA encoded a protein with a deduced molecular mass of91.7 kD that had features found in several known growth factorreceptors. It contained a single-membrane spanning region, an NH₂-terminal hydrophobic signal sequence, three extracellularimmunoglobulin-like domains and an intracellular tyrosine kinase domain(FIG. 6). Eleven potential N-linked glycosylation sites were also found.N- and O-linked glycosylation of the chicken bFGF receptor may accountfor the disparity between the observed size of the bFGF receptor and thesize predicted from the cDNA sequence.

Three immunoglobulin-like domains in the putative extracellular regionwere identified on the basis of three criteria: (i) the presence of twocharacteristic cysteine residues in each domain; (ii) the presence of aconsensus tryptophan residue 11 to 12 amino acids on the COOH-terminalside of the first cysteine residue in each immunoglobulin-like domain;and (iii) the presence of the consensus sequence, DXGXYXC, on theNH₂-terminal side of the second cysteine residue in eachimmunoglobulin-like domain. The interleukin-1 (IL-1) receptor also hasthree immunoglobulin-like domains, and bFGF has 25-30% sequence identityto IL-1. Five immunoglobulin-like domains are present in the receptorsfor platelet-derived growth factor (PDGF) and colony-stimulatingfactor-1 (CSF-1).

Between the first and second immunoglobulin-like domains, the bFGFreceptor has a feature not found in other members of the immunoglobulinsuperfamily. There is a series of eight consecutive acidic residues(EDDDDEDD) followed by three serine residues and two additional acidicresidues (FIG. 3). Although uninterrupted stretches of 7 to 35 acidicresidues have been described for several intracellular proteins, inparticular nuclear proteins, such acidic regions are unusual in theextracellular region of transmembrane receptor proteins.

Another unusual feature is the length of the juxtamembrane region, theregion between the membrane spanning segment and the kinase domain. Thisregion is normally conserved among receptor tyrosine kinases. Forexample, the juxtamembrane region is consistently 49 to 51 residues inlength in the receptors for PDGF, CSF-1, epidermal growth factor (EGF),human epidermal growth factor-2 (HER2) and insulin. The bFGF receptorhas an unusually long juxtamembrane region of about 87 residues.

The cytoplasmic region of the amino acid sequence is about 424 residueslong and contains a tyrosine kinase sequence (about residues 482 to759). Overall, the kinase region of the bFGF receptor shares the mostsequence identity (about 51 to 53%) with the PDGF and CSF-1 receptors.The bFGF receptor contains the GXGXXG motif and the conserved lysineresidue (about residue 512) that form part of the adenosine5′-triphosphate (ATP) binding site of tyrosine kinases. The bFGFreceptor also contains the two characteristic tyrosine kinase motifs,HRDLAARNVL and DFGLAR, and a tyrosine (about residue 651) at theposition analogous to the major phosphorylation site of pp60^(v−src)(about Tyr 416).

The kinase coding sequence of the bFGF receptor, defined by homology toother tyrosine kinases, is split by an insertion of 14 amino acids. Thelength of the insertion in the kinase region is shorter than that foundin the receptors for PDGF and CSF-1 (104 and 70 amino acids,respectively) and is similar to the length of the inserted sequence inthe receptors for insulin and insulin-like growth factor-I.

EXAMPLE 3 Full Lenath Human FGF Receptor cDNA Clone Preparation

A human FGF receptor cDNA clone was isolated from a human endothelialcell CDNA library obtained from E. Sadler (R. D. Ye T-C Wun & J. E.Sadler, J. Biol. Chem., 262: 3718-3725 (1987)) using the sameoligonucleotide probe described in Example 2.

The endothelial library was hybridized at high stringency with labeledprobe 1×10⁶ cpm/ml (50% formamide, 5×SSC, 5×Denhardts, 10 mM NaPO₄, pH6.5, 100 μg/ml salmon sperm DNA at 42° C., (16-24 hrs) and washed at 65°C. with 0.2×SSC, 0.1% SDS.

From the initial screening of the human endothelial cell cDNA library,four clones were identified and purified through 3 rounds of plaquepurification. The cDNA inserts from three of these clones generatedidentical sequences and contained sequences highly homologous to thesequences of tryptic fragments from the purified chicken bFGF-R. Theamino acid and nucleic acid sequence of the largest clone (approximately3.6 kb) is set forth in FIG. 4. Amino acids about 1-21 represent thehydrophobic signal sequence, about 22-285 the extracellular regioncontaining the ligand-binding domain, about 286-306 the transmembraneregion and about 307-731 the cytoplasmic region containing tyrosinekinase domain. This method also isolated other highly related human FGFreceptors.

EXAMPLE 4 Human aFGF-R cDNA Clone Preparation

Human endothelial cell or placental libraries are screened withfull-length FGF-R probes or probes containing a portion of the sequencefor FGF-R. Hybridization is performed at low stringency conditions andwashed in increments of increasingly higher stringency. The low and highstringency conditions described in Examples 2 and 3 are followed.Between each increment, autoradiography is performed. Clones which arepositive through to the most stringent conditions are most related tothe bFGF receptors previously described in Examples 2 and 3. Cloneswhich are positive at relaxed stringency but are no longer positive athigh stringency conditions are more distantly related. All related butnot identical (to FIG. 4) clones are determined by restriction mappingand DNA sequencing. All related clones are selected, subcloned andexpressed. The expressed FGF-related cDNAs are then tested for theirability to bind the various FGFS, i.e. acidic FGF.

Alternatively, two probes are designed, one probe containingintracellular FGF-R sequence and the other extracellular FGF-R sequence.Triplicate filters are made. One filter is hybridized at high stringency(see Examples 2 and 3) with the intracellular FGF-R probe. Two filtersare hybridized with the extracellular probe, one filter at highstringency and one at low stringency. Since acidic and basic FGFs haveonly 55% sequence identity, their receptors may also exhibit about 55%sequence identity in the ligand-binding domain. Clones which arepositive at high stringency to the intracellular probe and positive onlyat low stringency to the extracellular probe are FGF-R relatedreceptors. Thus clones are selected, restriction mapping performed,sequenced, and expressed. The expressed receptors are tested for theirability to bind to various FGFs, e.g., acidic FGF.

EXAMPLE 5 Characterization of Human FGF-R cDNA Clones

Plasmid Constructions.

For transfection experiments, full-length chicken FGF receptor cDNAcontaining 46 nucleotides of 5′ nontranslated sequence and the entire 3′nontranslated sequence, and full-length human h2 cDNA containing 13nucleotides of 5′ nontranslated sequence and the entire 3′ nontranslatedsequence were individually subcloned into the BamHI/SalI sites of themammalian expression vector pSV7d (P. Luciw, Chiron Corporation). Thisplaced the receptor cDNA fragments in the proper orientation directlydownstream from an SV40 promoter element.

To prepare constructs to be used as templates for generating in vitrotranscribed RNAs, full-length chicken FGF receptor cDNA was subclonedinto the BamHI/SalI sites of Bluescript Sk (Stratagene) and full-lengthhuman FGF receptor cDNAs (h2 and h3) were subcloned into the PstI/SalIsites of Bluescript KS. This placed the receptor sequences directlydownstream from the T7 RNA polymerase promoter element. To enhance thepossibility of efficient translation, ATG sequences upstream of theinitiator methionine residue were removed prior to subcloning, leaving46 and 13 nucleotides of intact 5′ nontranslated sequence for thechicken and human constructs, respectively.

Cell Lines and Transfections.

Rat L6 skeletal muscle myoblasts (ATCC CRL 1458) were grown in DME H21containing 10% fetal calf serum and transferred into opti-MEM (GIBCO)just prior to transfection. Within 24 hours after plating, 1×10⁶ cellswere cotransfected with 20 μg of the appropriate expression construct(either cFGFR/pSV7d or h2FGFR/pSV7d) and 1 μg of a vector containing theneomycin resistance gene (pSV2neo). Cells were transfected using 50 μgof Lipofectin (Bethesda Research Laboratories) following the protocolprovided by the manufacturer. Sixteen hours later, an equal volume ofDHE H21 media containing 20% fetal calf serum was added. After 48 hours,cells were harvested and passaged (1:10) into selection media (DME H21,10% fetal calf serum, 500 μg/ml geneticin (GIBCO). Transfectant colonieswere assayed for expression of the FGF receptor by immunoblotting withanti-receptor peptide polyclonal antisera.

Affinity Labeling.

Recombinant human aFGF and human bFGF were generously donated by ChironCorporation and indicated. For affinity labeling experiments, 5×10⁶cells were incubated for 30 minutes at 37° C. with 0.1 pmoles of¹²⁵I-aFGF or ¹²⁵1-bFGF in the presence or absence of a 200-fold excessof the corresponding unlabeled ligand. The cells were then washed oncewith ice cold DME H21 containing 20 mM HEPES pH 7.4, 0.2% gelatin, andtwice with ice cold PBS. Disuccinimidyl suberate was added to a finalconcentration of 0.15 mM and incubations were allowed to proceed for 15minutes at 4° C. The crosslinking agent was then removed and the cellswere resuspended in sample buffer containing 100 mM dithiothreitol,boiled for 5 minutes, and subjected to SDS PAGE followed byautoradiography.

In vitro Transcription of RNA.

Prior to transcription, plasmid constructs were linearized with XhoI.RNAs were transcribed from the linearized templates using T7 RNApolymerase in the presence of 500 μM rNTPs (200 μM rGTP) and 500 μM^(5′) GpppG³, (Pharmacia). Following incubation at 4° C. for 2 hours,transcription reactions were treated with RNAse-free DNAse, phenolextracted, ethanol precipitated, dried and resuspended in water.

Injection of Oocytes.

Animals were anesthetized in a solution of 0.06 percent ethylp-aminobenzoate. Oocytes were surgically removed and manually dissectedinto clusters containing 10-20 oocytes. Clusters were incubated inmodified Barth Saline (See Maniatis, et al., Molecular Cloning: ALaboratory Manual, CSH Press (1982), which is incorporated herein byreference.) MBSH containing 1 mg/ml Type II collagenase (Sigma) for 2hours at room temperature and then washed extensively with MBSHcontaining 2 mg/ml bovine serum albumin (BSA). Individual oocytes weremaintained at 19° C. in MBSH (1 mg/ml BSA).

Oocytes were injected into the vegetal pole with 50 nl of water or RNAsolution (1 μg/μl in water). Following injection, oocytes were incubatedat 9° C. for 48 hours before performing ⁴⁵Ca⁺⁺efflux assays.

⁴⁵Ca⁺⁺Efflux Assays.

Groups of 50 injected oocytes were added to single wells of a 24 wellplate and washed four times with 0.5 ml of a Ca⁺⁺-free MBSH solutioncontaining no BSA. Oocytes were then incubated in 0.5 ml of the washsolution containing ⁴⁵CaCl₂ (100 μCi/ml) for 3 hours at 19° C. Followingincubation, oocytes were washed six times with 0.5 ml of MBSH (1 mg/mlBSA), then transferred to another 24 well plate (5 oocytes per well).All subsequent washes and incubations were performed using 0.5 ml ofMBSH containing 1 mg/ml BSA. At 10 minute time intervals, conditionedsupernatants were removed from each well and replaced with fresh media.The conditioned media samples were counted individually in a Beckmanscintillation counter. When background efflux stabilized, ligands wereadded to the specified concentrations and media collections werecontinued.

In 2 out of 16 experiments, oocytes injected with water and stimulatedwith either aFGF or bFGF exhibited ⁴⁵Ca⁺⁺ efflux levels similar to thoseobtained from oocytes injected with FGF receptor RNA. We have notdetermined the reason for these unexpected responses, but it is possiblethat they were due to expression of endogenous FGF receptors oncontaminating follicular cells, or on the surface of the oocytesthemselves. In all other experiments the water injected oocytes had nosignificant efflux response whereas the receptor RNA response to FGF wasten to forty fold over the basal measurement.

Receptor levels in injected oocytes have not been measured because ouranti-receptor polyclonal antisera nonspecifically recognizes an abundantoocyte protein of approximately the same molecular weight as the FGFreceptor on western blots. Furthermore, the levels of exogenousreceptors expressed in oocytes appears to be quite low.

Isolation and Characterization of Human cDNA Clones.

Complementary DNA libraries from human placenta and human umbilical veinendothelial cells were generously donated by J. Evan Sadler (WashingtonUniversity School of Medicine, St. Louis). The libraries were screenedwith ³²P-labeled oligomers identical to those previously used toidentify chicken FGF receptor cDNA clones. Filters were hybridized andwashed under high stringency conditions using standard methods. A totalof 7 positive clones were isolated after screening 250,000 phage fromboth libraries. The 4 clones described in this report (h2, h3, h4, andh5) were sequenced by the dideoxy chain termination method, using theSequenase system (United States Biochemical Corporation). Clones h2, h3,and h4 were obtained from the endothelial cell library and clone h5 wasobtained from the placenta library. Nucleotide sequence analysesrevealed that all four clones contained identical 5′ nontranslatedsequences and had poly-A tracts at their 3′ ends. However, only thepoly-A tract at the 3′ end of h2 was preceded upstream by a consensuspoly adenylation signal sequence (AATAAA: 37), indicating that internalpriming was responsible for the poly A tracts at the 3′ ends of theother clones. The h2, h3, h4, and h5 cDNAs contained 0.93 kb, 0.78 kb,0.95 kb, and 0.2 kb of 3′ nontranslated sequence, respectively. The 3′nontranslated sequences of h2 and h3 were identical and the 3′nontranslated sequences of h4 and h5 were also identical. In contrast,the h2/h3′ nontranslated sequences were entirely different from the 3′nontranslated sequences of h4/h5.

Polymerase Chain Reactions.

Amplification reactions (42) were carried out using one primercorresponding to the human highly acidic region (approximately aminoacids 44-52 in h2; S′GTTTCTTCTCCTCTGAAGAGGAGT-3′) and one degenerateprimer corresponding to the IgI domain of the chicken FGF receptor(approximately amino acids 58-69; 5′- GA(TC)GACGTGCAG(A/T)(G/C)CATCAACTGGGTGCGTGATGG-3′). In additional reactions, we usedthe primer from the human highly acidic region and a second primerderived from the 5′ nontranslated region of the human FGF receptor(5′-GAGGATCGAGCTCACTGTGGAGTA-3′). Reaction mixtures contained 750 ng ofhuman genomic DNA, 10 pmoles of each primer, 200 μM of each of the fourdNTPs, and 1 unit of Taq polymerase (Perkin Elmer Cetus) in 50 μl of 10mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 100 ng/ml BSA. Reactionswere carried out in an Ericomp twin block system. Thirty one cycles wereperformed, consisting of denaturation at 94°C. for 50 seconds, annealingat 65°C. for 1 minute, and extension at 72° C. for 3 minutes.

Isolation and Characterization of Four Unique Human FGF Receptor cDNAs.

The chicken basic FGF receptor contains a single transmembrane domain,an extracellular region containing 3 Ig-like domains and a highly acidicdomain, and an intracellular region containing a split tyrosine kinasedomain. The chicken FGF receptor cDNA is highly homologous to apreviously published partial cDNA (hfla) which encodes a tyrosine kinasethat, at the time of its description was of unknown function. The highdegree of identity (95 percent) between the chicken bFGF receptor andhuman flg suggested that hflg was the human counterpart of the bFGFreceptor. To obtain full-length human FGF receptor cDNAs,oligonucleotide probe based on the hflg DNA sequence was used to screena human umbilical vein endothelial cell cDNA library and a humanplacenta cDNA library. From the initial screenings of 250,000 plaquesfrom each library, four positive clones were isolated from theendothelial cell library and three from the placenta library.

The CDNA clones could be divided into two classes based on differentpatterns of restriction maps at their 3′ ends. One of these classesderived from cDNA clones which were much shorter in length.Representatives of each class were present in the clones isolated fromeither library. Two clones (h2 and h3) representing the class of largercDNA clones, and two clones (h4 and h5) representing the class ofshorter cDNA clones were sequenced in their entirety. The deduced aminoacid sequences of the four human receptor forms are shown in comparisonto the chicken FGF receptor sequence in FIG. 7. A schematicrepresentation of the different receptor forms is shown in FIG. 8.

The predicted amino acid sequences of the h2 and h3 clones are virtuallyidentical and differ only by three amino acids (amino acids 59, 60, and103 in h2, FIG. 7). At the nucleotide level, h2 and h3 differ only atthe positions encoding these three amino acid residues. The h2/h3 openreading frames include a hydrophobic signal sequence and the unusualacidic domain (8 consecutive acidic residues with accompanying residues)that was initially noted in the published sequence of the chicken FGFreceptor cDNA. The extracellular domains of h2 and h3 are highlyhomologous to the chicken FGF receptor except that h2 and h3 lack thesequences of one Ig-like domain (labeled I in FIG. 8). The transmembraneregion and cytoplasmic domains are highly homologous to thecorresponding domains of the chicken FGF receptor.

The coding sequences of the short cDNA clones, h4 and h5, differ only bytwo amino acids (positions 59 and 60 in h4; the nucleotide sequences ofh4 and h5 differ only at the positions encoding these two residues). Thesignal sequence, acidic region and one of the Ig-like domains (IgII) areessentially identical to the corresponding regions of h2 and h3. Thedistinctive feature of h4 and h5 is the Ig-like domain (IgIII) nearestthe transmembrane domain. Approximately half of this domain is identicalto the corresponding sequence of h2 and h3. However, the carboxylterminal half of this Ig-like domain is unrelated to h2 and h3sequences. Unlike h2, h3, and the chicken FGF receptor DNA, h4 and h5 donot encode a hydrophobic membrane spanning region or a cytoplasmicdomain.

The sequences of all of the human cDNAs which have been isolated containonly 2 Ig-like domains. To determine whether the human FGF receptor genecontains sequences encoding the first Ig-like domain (IgI), polymerasechain reactions were performed on genomic DNA isolated from humanforeskin fibroblasts (HFFs). For these experiments, we utilized oneamplifying primer based on the sequence of the IgI domain of the chickenreceptor (corresponding to amino acids 58-69), and a second primer basedon sequence from the acidic region of the human receptor (amino acids44-52 in h2). Using these primers, a single 1.3 kb genomic fragment wasamplified. As shown in FIG. 9, this fragment contained coding sequenceshomologous (approximately 83 percent amino acid identity) to the IgIdomain of the chicken FGF receptor. In addition, an intron sequence ofapproximately 1.0 kb separates these coding sequences from sequencesencoding the highly acidic region of the receptor. Thus, the human FGFreceptor gene clearly contains sequences encoding the IgI domain notfound in the human cDNA clones. Furthermore, the presence of an intronbetween the IgI domain sequence and the acidic region sequence suggeststhat expression of 2 or 3 Ig domain forms may be regulated byalternative splicing.

To determine whether a 3 Ig domain form of the receptor is expressed inHFF cells, we performed PCR on cDNA generated from HFF mRNA. Using theprimers described above, a single 0.24 kb fragment was amplified fromHFF DNA. This fragment contained sequences encoding the IgI domain andthe acidic region, but no intron sequences. Thus, we conclude that HFFcells transcribe a 3 Ig domain form of the receptor. To determinewhether HFF cells also express a 2 Ig domain form of the receptor, weutilized the acidic region primer and a second primer based on sequencefrom the 5′ nontranslated region of the human FGF receptor. In thesereactions a 0.23 kb fragment was amplified which, in the same manner asour cDNA clones, was missing sequences corresponding to the IgI domain.Thus, a 2 Ig domain form of the receptor is also transcribed in HFFcells.

Receptors Containing 3 Ig-like and 2 Ig-like Domains Bind Acidic FGF andBasic FGF.

Since the 3 Ig domain receptor (initially isolated from a chicken cDNAlibrary) was purified on the basis of its affinity for basic FGF, it wasof interest to determine whether this receptor also binds acidic FGF. Toaddress this question, the 3 Ig domain chicken receptor was expressed inrat L6 myoblasts, a cell line which normally does not express FGFreceptors. In addition, the 2 Ig domain human h2 receptor was alsoexpressed in L6 cells. FIG. 10 shows an affinity labeling experimentperformed with transfected cells. Cells were incubated with either¹²⁵I-aFGF or ¹²⁵I-bFGF and bound ligand was crosslinked in the presenceof disuccinimidyl suberate (0.15 mM). Using either ligand, singlecrosslinked bands were seen in cells transfected with receptor cDNAs(lanes 1, 3, 7, and 9), but not in cells transfected with vector alone(lanes 5, 6, 11, and 12). Subtraction of the molecular weight of FGF (17kd) from the size of the crosslinked complexes yields estimatedmolecular weights of 145 kd for the 3 Ig domain form of the receptor and125 kd for the 2 Ig domain form of the receptor. Excess unlabeledligands block formation of the crosslinked complexes (lanes 2, 4, 8, and10). These results demonstrate that both the 3 Ig domain form and the 2Ig domain form of the FGF receptor are capable of binding either acidicor basic FGF. Scatchard binding analyses indicate that half-maximalbinding of ¹²⁵I-aFGF to either the 3 Ig domain form or the 2 Ig domainform occurs at a concentration of 0.05 nM. Similarly, half-maximalbinding of ¹²⁵I-bFGF to either the 3 Ig domain form or the 2 Ig domainform occurs at 0.1 nM.

A Three Ig Domain FGF Receptor and a Two Ig Domain FGF Receptor MediateBiological Responses to Both Acidic and Basic FGF.

To determine whether any of the membrane spanning forms of the FGFreceptor are activated by either aFGF or bFGF, we expressed thesereceptors in Xenopus oocytes and measured receptor activation using asensitive Ca⁺⁺ efflux assay. This assay has been used to examineexpression of receptors for other Ca⁺⁺ mobilizing ligands includingcholecystokinin, bombesin, vasopressin, and angiotensin II.Ligand-induced efflux reflects a mobilization of Ca⁺⁺ from intracellularstores, leading to increased levels of intracellular Ca⁺⁺ andaccelerated efflux. For our experiments full-length cDNA weretranscribed in vitro and the capped mRNAs were injected into Xenopusoocytes. After 48 hours, the injected oocytes were loaded with ⁴⁵CaCl₂and ligand-dependent calcium mobilization was assayed by measuring⁴⁵Ca⁺⁺ efflux (FIG. 11). Addition of either aFGF (A and B) or bFGF (Cand D) induced a rapid and large efflux of ⁴⁵Ca⁺⁺ from oocytes injectedwith RNA encoding the chicken FGF receptor (A and C) or RNA encoding thehuman h2 receptor (B and D). In contrast, oocytes injected with eitherhuman h3 RNA (B and D) or water alone (A-D) showed no response to eitheraFGF or bFGF. As a positive control, carbachol was added following the100 minute timepoint. Oocytes express endogenous receptors forcarbachol, and oocytes injected with either FGF receptor RNA or waterexhibited a positive response after carbachol stimulation. We concludethat both the 3 Ig domain form (cFGF-R) and the 2 Ig domain form (h2) ofthe FGF receptor are biologically responsive to both acidic and basicFGF. Thus, the ligand binding domains for acidic and basic FGF appear tolie in the receptor region encompassing the highly acidic domain and theIgII and IgIII domains.

While the human h2 receptor clearly responds to both ligands, noresponse was seen in oocytes injected with RNA encoding the h3 receptorform. It is possible that the three amino acid differences between h2and h3 cause these proteins to respond differently. Alternatively, thelack of a response in oocytes injected with the h3 RNA may be due tounusually low expression levels of the h3 protein. Unfortunately, wehave not yet been able to determine receptor protein expression levelsin oocytes.

FGF-R forms having either 2 or 3 extracellular Ig-like domains will bindand respond to both acidic and basic FGF. Some forms of FGF receptormRNA encode only the extracellular domain of the FGF receptor, a proteinthat is likely to be secreted from the cell.

The fact that a 2 Ig-like domain form of the FGF receptor (h2) bindsboth aFGF and bFGF with high affinity has allowed us to localize thebinding domains for these ligands to a region encompassing the highlyacidic region and the IgII and IgIII domains.

The h4 and h5 receptor forms lack transmembrane sequences and presumablyrepresent secreted forms of the FGF receptor. Preliminary data indicatesthat cells transfected with the h4 cDNA secrete a 70 kd protein which isrecognized by anti-FGF-R polyclonal antisera.

The role of secreted forms of the FGF receptor is unclear. The secretedforms may act to regulate levels of extracellular FGFs, and therebyregulate availability of FGFs to cell surface FGF receptors.Alternatively, the secreted FGF receptors may serve to store andsequester FGFs at a particular location. Another possibility is that thesecreted forms may bind to FGFs in an intracellular compartment andsubsequently serve as a means for secreting the factor. This is animportant consideration in view of the fact that aFGF and bFGF do notcontain signal sequences and their mechanism of secretion is unknown.

our results suggest that receptor diversity can be generated byalternative splicing. We have isolated a total of 5 different FGFreceptor cDNA species. Comparison of amino acid sequences stronglyindicates that all 5 species are derived from the same gene. Anotherinteresting feature of the human receptor forms is the presence orabsence of the ArgMet sequence (amino acids 59 and 60 in h2 and h4) inthe extracellular domain.

Affinity labeling experiments using either ¹²⁵I-aFGF or ¹²⁵I-bFGFidentified a single 145 kd receptor protein on transfectant cellsexpressing the 3 Ig domain form of the FGF receptor, and a single 125 kdreceptor protein on transfectant cells expressing the 2 domain form ofthe FGF receptor (see FIG. 11). It is possible that the presence of tworeceptor species may reflect coexpression of the 3 Ig domain and 2 Igdomain forms of the receptor. Our data clearly establish that a singleFGF receptor species can bind both aFGF and bFGF with high affinity andmediate the biological effects of these factors. We have used acidic andbasic FGF in these experiments because they are the best characterizedmembers of the FGF family, and are readily available in recombinantform.

EXAMPLE 6 Competitive Binding of FGF-R Peptides or Fragments Developmentof FGF-R Related Antagonists or Agonists

A fragment containing all or part of the extracellular, ligand-bindingdomain of the FGF-R (i.e., containing amino acids 22-374 of FIG. 3 or22-285 of FIG. 4) or analogs thereof are expressed in a host (e.g.mammalian cells or baculovirus infected insect cells) and purified asdescribed in Example 1. Alternatively, fragments of the ligand-bindingdomain are made using a peptide synthesizer (Applied Biosystems) andpurified by HPLC. Different concentrations of the FGF-R fragment oranalogs thereof (FGF-Rexs) are tested for their ability to block thebinding of ¹²⁵I-FGF to Swiss 3T3 cells. Competitive binding is performedas described in FIG. 1A in Example 1 using FGF-Rexs instead of unlabeledligand and competitive binding is determined.

FGF-Rexs are also tested for their ability to inhibit FGF-inducedmitogenesis as measured by ³H-thymidine incorporation into cells and bycounting cell numbers. FGF-Rexs which block binding of FGF to thecell-surface receptor may act as an antagonist and block ³H-thymidineuptake and the increase in cell number induced by FGF. FGF-Rexs may alsoact as agonists, i.e. by dimerization with the cell surface receptorwhich may mimic a ligand-mediated receptor-recaptor interaction. In suchan instance, FGF-Rexs may stimulate mitogenesis in the absence of ligandor may enhance the FGF mediated mitogenic response.

FGF-Rexs are also tested for their ability to inhibit or activateFGF-induced tyrosine phosphorylation of the 90 substrate protein inSwiss 3T3 cells or autophosphorylation of the cell-associated FGF-R.FGF-Rexs which block FGF-induced tyrosine phosphorylation areantagonists. FGF-Rexs which activate autophosphorylation of thecell-associated FGF-R in the absence of FGF are agonists.

FGF-Rexs are also tested for their anti-angiogenic activity. FGF-Rexsare tested first for their ability to inhibit the FGF-induced growth andthe mobilization of endothelial cells into vessels in vitro.Angiogenesis is assayed in vitro using an aortic ring assay. Aorticrings are placed in a collagen matrix formed in the presence or absenceof FGF and FGF-Rexs. Endothelial cells sprout and form vessels from theaortic ring within a few days in the presence of FGF. The addition ofFGF-Rexs which are antagonists in the previous assays inhibit theFGF-induced growth of capillary sprouts. FGF-Rexs which are angiogeniceven in the absence of FGF are agonists.

FGF analogs, angiogenic factors, anti-angiogenic factors as well asantibodies to the extracellular portion of the FGF-R are tested fortheir ability to bind directly or compete for binding of native FGF forbinding to purified or expressed FGF-R. In addition, they are tested fortheir ability to stimulate mitogenesis (agonists) or inhibitFGF-dependent mitogenesis (antagonists) as well as tyrosinephosphorylation in cells expressing the FGF-R. These studies areimportant in determining if the mode of action of each angiogenic andanti-angiogenic factor, etc., is receptor-mediated and in determining ifthere is receptor specificity (i.e. acidic versus basic FGF-R) forangiogenic and anti-angiogenic factors.

FGF analogs are radiolabeled and binding is performed with labeledligand, purified or expressed receptor in the appropriate physiologicbuffer (i.e. culture media or phosphate buffered saline (PBS)) for 0.5at 37° C. or 2-24 hrs at 4° C. The complex is precipitated (5-10%polyethylene glycol, 1 mg/ml IgG) and separated by filtration throughfilters (i.e. Whitman GFA) and the associated radioactivity determined.

While the invention has been described in connection with certainspecific embodiments thereof, it should be recognized that variousmodifications as may be apparent to one of skill in the art to which theinvention pertains also fall within the scope of the invention asdefined by the appended claims.

What is claimed is:
 1. An isolated nucleic acid encoding a segment of atleast seven contiguous amino acids of a human fibroblast growth factorreceptor shown in FIGS. 7A and 7B, which segment lacks a tyrosine kinasedomain, wherein the segment can specifically bind to an antibody to thehuman fibroblast growth factor receptor.
 2. An isolated nucleic acid ofclaim 1, wherein said contiguous amino acids are from an IgII domain ofa human fibroblast growth factor receptor described in FIG.
 7. 3. Anisolated nucleic acid of claim 1, encoding a full-length IgII domain ofa fibroblast growth factor receptor shown in FIGS. 7A and 7B.
 4. Anisolated nucleic acid of claim 3, further encoding at least oneadditional peptide segment from a fibroblast growth factor receptorshown in FIGS. 7A and 7B selected from the group consisting of a signalpeptide, an IgI domain, an acidic segment, an IgIII segment, an IgIIITsegment, and a transmembrane segment.
 5. An isolated nucleic acid ofclaim 3, further comprising a transcription promoter sequence.
 6. Theisolated nucleic acid of claim 1, which encodes at least 13 contiguousamino acids from a fibroblast growth factor receptor shown in FIGS. 7Aand 7B.
 7. The isolated nucleic acid of claim 6, which encodes at least20 contiguous amino acids from a fibroblast growth factor shown in FIGS.7A and 7B.
 8. The isolated nucleic acid of claim 1, wherein the segmentcomprises at least seven contiguous amino acids from the humanfibroblast growth factor receptor h3 of FIG. 7A and 7B.
 9. An isolatednucleic acid that hybridizes under stringent conditions including 50%formamide, 5×SSC, 5×Denhardt's solution, 10 mM sodium phosphate, pH 6.5,100 μg/ml salmon sperm DNA and at 42° C. to a segment of a nucleic acid,which segment encodes an IgII domain of a soluble fibroblast growthfactor receptor shown in FIGS. 7A and 7B, the isolated nucleic acidlacking a segment encoding a tyrosine kinase domain and wherein the IgIIdomain can specifically bind to an antibody to the soluble fibroblastgrowth factor receptor.
 10. An isolated nucleic acid of claim 9, whereinsaid soluble growth factor receptor is h4 or h5.
 11. A nucleic acidaccording to claim 1, 3, or 9, attached to a reporter molecule.
 12. Anisolated nucleic acid encoding the human fibroblast growth factorreceptor designated h3 in FIGS. 7A and 7B.
 13. An isolated nucleic acidencoding the human fibroblast growth factor receptor designated h2 inFIGS. 7A and 7B.
 14. An isolated cell line transformed with a DNAsequence capable of expressing a polypeptide comprising at least sevencontiguous amino acids of an extracellular domain of a fibroblast growthfactor receptor shown in FIGS. 7A and 7B, the polypeptide lacking atyrosine kinase domain and wherein the polypeptide can specifically bindto an antibody to the human fibroblast growth factor receptor.
 15. Theisolated cell line of claim 14, wherein the polypeptide comprises asoluble form of a human fibroblast growth factor receptor.
 16. Anisolated cell line as in claim 15, wherein the cell is capable ofsecreting the human fibroblast growth factor receptor.
 17. The isolatedcell line of claim 14, wherein the polypeptide comprises at least 13contiguous amino acids of a fibroblast growth factor receptor shown inFIGS. 7A and 7B.
 18. The isolated cell line of claim 14, wherein thepolypeptide comprises at least 20 contiguous amino acids of a fibroblastgrowth factor receptor shown in FIGS. 7A and 7B.
 19. The isolated cellline of claim 14, wherein the polypeptide comprises at least sevencontiguous amino acids of an extracellular domain of the fibroblastgrowth factor receptor h3 shown in FIGS. 7A and 7B.
 20. A method ofmaking a protein comprising a fibroblast growth factor receptor segment,said method comprising expressing an isolated nucleic acid encoding asegment of at least seven contiguous amino acids of a fibroblast growthfactor receptor shown in FIGS. 7A and 7B, which segment lacks a tyrosinekinase domain and wherein the segment can specifically bind to anantibody to the human fibroblast growth factor receptor, and recoveringthe segment.
 21. The method of claim 20, wherein the isolated nucleicacid encodes at least 13 contiguous amino acids from a fibroblast growthfactor receptor shown in FIGS. 7A and 7B.
 22. The method of claim 21,wherein the isolated nucleic acid encodes at least 20 contiguous aminoacids from a fibroblast growth factor receptor shown in FIGS. 7A and 7B.23. The method of claim 20, wherein the segment comprises at least 7contiguous amino acids from the fibroblast growth factor receptor h3 ofFIGS. 7A and 7B.
 24. A method of making a fibroblast growth factorreceptor or peptide thereof, the method comprising expressing a nucleicacid in a host cell to produce the fibroblast growth factor receptor orpeptide and recovering the fibroblast growth factor receptor or peptidefrom the host cell or media containing the host cell, wherein thenucleic acid comprises at least 21 bases of a sequence selected from thegroup consisting of: a DNA sequence in FIG. 3, a DNA sequence in FIGS.4A, B, C and D, a DNA sequence in FIG. 9, and a DNA sequence encoding atleast seven contiguous amino acids of a polypeptide of FIGS. 7A and B;wherein the fibroblast growth factor receptor or peptide comprises anextracellular domain and lacks a tyrosine kinase domain and wherein thereceptor or peptide can specifically bind to an antibody to the humanfibroblast growth factor receptor.
 25. A method of making a proteincomprising a fibroblast growth factor receptor segment, said methodcomprising expressing an isolated nucleic acid encoding a full-lengthIgII domain of a fibroblast growth factor receptor shown in FIGS. 7A and7B to produce the fibroblast growth factor receptor segment and whereinthe full-length IgII domain can specifically bind to an antibody to thehuman fibroblast growth factor receptor, and recovering the fibroblastgrowth factor receptor segment.
 26. A method of forming a compositioncomprising a fibroblast growth factor segment, comprising: expressing anisolated nucleic acid encoding a segment of at least seven contiguousamino acids of a fibroblast growth factor receptor shown in FIGS. 7A and7B, which segment lacks a tyrosine kinase domain, and wherein thesegment can specifically bind to an antibody to the human fibroblastgrowth factor receptor and recovering the segment; and combining thesegment with a carrier as a composition.